Anti-notch2 nrr antibodies

ABSTRACT

The invention provides anti-Notch antibodies, and in particular, antibodies that bind Notch2 NRR, and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/298,488, filed Jun. 6, 2014, which is a continuation of U.S. patentapplication Ser. No. 14/051,699, filed Oct. 11, 2013, now abandoned,which is a continuation of U.S. patent application Ser. No. 13/773,843,filed Feb. 22, 2013, now abandoned, which is a continuation of U.S.application Ser. No. 12/570,267, filed Sep. 30, 2009, now U.S. Pat. No.8,404,239, which claims the benefit of U.S. Provisional Application No.61/101,917, filed Oct. 1, 2008, the disclosures of each of which arehereby incorporated by reference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 9, 2016, isnamed 2017-07-21_01146-0009-04US_SeqList_ST25.txt and is 67,062 bytes insize.

FIELD OF THE INVENTION

The present invention relates generally to the field of molecularbiology. More specifically, the invention concerns anti-Notchantibodies, including anti-Notch2 negative regulatory region (NRR)antibodies, and uses of the same. Anti-Notch1 NRR antibodies and methodsof use are also provided.

BACKGROUND

The Notch receptor family is a class of evolutionarily conservedtransmembrane receptors that transmit signals affecting development inorganisms as diverse as sea urchins and humans. Notch receptors andtheir ligands, Delta and Serrate (known as Jagged in mammals), aretransmembrane proteins with large extracellular domains that containepidermal growth factor (EGF)-like repeats. The number of Notchparalogues differs between species. For example, there are four Notchreceptors in mammals (Notch1-Notch4), two in Caenorhabditis elegans(LIN-12 and GLP-1) and one in Drosophila melanogaster (Notch). Notchreceptors are proteolytically processed during transport to the cellsurface by a furin-like protease at a site S1 on the N-terminal side ofthe transmembrane domain, producing an extracellular Notch (ECN) subunitand a Notch transmembrane subunit (NTM). These two subunits remainnon-covalently associated and constitute the mature heterodimericcell-surface receptor. Notch receptors and the Notch signaling pathwayare reviewed, e.g., in Aster et al., Annu. Rev. Pathol. Mech. Dis.3:587-613, 2008, and Bolos et al., Endocrine Reviews 28:339-363, 2007.

Notch2 ECN subunits contain 36 N-terminal EGF-like repeats followed bythree tandemly repeated Lin 12/Notch Repeat (LNR) modules that precedethe S1 site. Each LNR module contains three disulfide bonds and a groupof conserved acidic and polar residues predicted to coordinate a calciumion. Within the EGF repeat region lie binding sites for the activatingligands.

The Notch2 NTM comprises an extracellular region (which harbors the S2cleavage site), a transmembrane segment (which harbors the S3 cleavagesite), and a large intracellular portion that includes a RAM23 domain,six ankyrin repeats, a transactivation domain and a carboxy-terminalPEST sequence. Stable association of the ECN and NTM subunits isdependent on a heterodimerization domain (HD) comprising thecarboxy-terminal end of the ECN (termed HD-N) and the extracellularamino-terminal end of NTM (termed HD-C). Before ligand-inducedactivation, Notch is maintained in a resting conformation by a negativeregulatory region (NRR), which comprises the three LNRs and the HDdomain. The crystal structure of the Notch2 NRR is reported in Gordon etal., (2007) Nature Structural & Molecular Biology 14:295-300, 2007.

Binding of a Notch ligand to the ECN subunit initiates two successiveproteolytic cleavages that occur through regulated intramembraneproteolysis. The first cleavage by a metalloprotease (ADAM17) at site S2renders the Notch transmembrane subunit susceptible to a second cleavageat site S3 close to the inner leaflet of the plasma membrane. Site S3cleavage, which is catalyzed by a multiprotein complex containingpresenilin and nicastrin and promoting γ-secretase activity, liberatesthe intracellular portion of the Notch transmembrane subunit, allowingit to translocate to the nucleus and activate transcription of targetgenes. (For review of the proteolytic cleavage of Notch, see, e.g.,Sisodia et al., Nat. Rev. Neurosci. 3:281-290, 2002.)

Five Notch ligands of the Jagged and Delta-like classes have beenidentified in humans (Jagged1 (also termed Serrate1), Jagged2 (alsotermed Serrate2), Delta-like1 (also termed DLL1), Delta-like3 (alsotermed DLL3), and Delta-like4 (also termed DLL4)). Each of the ligandsis a single-pass transmembrane protein with a conserved N-terminalDelta, Serrate, LAG-2 (DSL) motif essential for binding Notch. A seriesof EGF-like modules C-terminal to the DSL motif precede themembrane-spanning segment. Unlike the Notch receptors, the ligands haveshort cytoplasmic tails of 70-215 amino acids at the C-terminus. Inaddition, other types of ligands have been reported (e.g., DNER, NB3,and F3/Contactin). (For review of Notch ligands and ligand-mediatedNotch activation, see, e.g., D'Souza et al., Oncogene 27:5148-5167,2008.)

The Notch pathway functions during diverse developmental andphysiological processes including those affecting neurogenesis in fliesand vertebrates. In general, Notch signaling is involved in lateralinhibition, lineage decisions, and the establishment of boundariesbetween groups of cells (see, e.g., Bray, Molecular Cell Biology7:678-679, 2006). A variety of human diseases, including cancers andneurodegenerative disorders, have been shown to result from mutations ingenes encoding Notch receptors or their ligands (see, e.g., Nam et al.,Curr. Opin. Chem. Biol. 6:501-509, 2002). The connection betweenunrestrained Notch signaling and malignancy was first recognized when arecurrent t(7;9)(q34;q34.3) chromosomal translocation which creates atruncated, constitutively active variant of human Notch1 was identifiedin a subset of human acute lymphoblastic leukemias (T-ALL) (see, e.g.,Aster et al., Annu. Rev. Pathol. Mech. Dis. 3:587-613, 2008). In mousemodels, Notch1 signaling has been shown to be essential for T celldevelopment and that Notch1-mediated signals promote T cell developmentat the expense of B cell development (see, e.g., Wilson et al., J. Exp.Med. 194:1003-1012, 2001).

Notch2 is also involved in certain cancers. Particularly, Notch2 isoverexpressed in B-cell chronic lymphocytic leukemia (B-CLL), which inturn leads to overexpression of CD23, a hallmark of B-CLL cells. (SeeHubmann et al., Blood 99:3742-3747, 2002.) Both Notch1 and Notch2 arehighly expressed in multiple myeloma cells (cancerous plasma B cells),and stimulation with ligand strongly increases tumor cell growth. (SeeJundt et al., Blood 103:3511-3515, 2004.) Notch2 and downstreameffectors are overexpressed in melanoma (see Hoek et al., Cancer Res.64:5270-5282, 2004; Seykora et al., Am J Dermatopathol 25:6-11, 2003),and the Notch2 locus is recurrently amplified in melanoma cell lines(Jonsson et al., Oncogene, 26:4738-4748, 2007). In addition, numerousstudies have linked aberrant Notch2 signaling to breast cancer and othersolid tumors (reviewed by Leong and Karsay, Blood 107:2223-2233, 2006).Notch2 is also required for marginal zone B cell development. (SeePillai et al., Annu. Rev. Immunol. 23:161-196, 2005.)

Given the involvement of Notch signaling in a wide variety of humandiseases it is clear that there continues to be a need for agents thatregulate Notch signaling and that have clinical attributes that arefavorable for development as therapeutic agents. The invention describedherein meets this need and provides other benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY

The invention provides anti-Notch antibodies and methods of using thesame.

In one aspect, a monoclonal antibody that binds to Notch2 NRR isprovided. In one embodiment, the antibody inhibits Notch2 activity. Inanother embodiment, the antibody does not significantly bind to a Notchfamily member other than Notch2. In another embodiment, the antibodybinds to mouse Notch2 NRR and human Notch2 NRR. In another embodiment,the antibody binds to a Notch2 NRR with a Kd of ≦10 nM.

In a further embodiment, a monoclonal antibody that binds to Notch2 NRRis provided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:3;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:10;    -   (e) an HVR-L2 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:14; and    -   (f) an HVR-L3 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:19.        In one such embodiment, the antibody comprises an HVR-H1        comprising an amino acid sequence selected from SEQ ID NOs:1-2;        an HVR-L1 comprising an amino acid sequence selected from SEQ ID        NOs:6-9; an HVR-L2 comprising an amino acid sequence selected        from SEQ ID NOs:11-13; and an HVR-L3 comprising an amino acid        sequence selected from SEQ ID NOs:15-18. In one such embodiment,        the HVR-H1 comprises the amino acid sequence of SEQ ID NO:1, the        HVR-L1 comprises the amino acid sequence of SEQ ID NO:6, the        HVR-L2 comprises the amino acid sequence of SEQ ID NO:11, and        the HVR-L3 comprises the amino acid sequence of SEQ ID NO:15. In        another such embodiment, the HVR-H1 comprises the amino acid        sequence of SEQ ID NO:2, the HVR-L1 comprises the amino acid        sequence of SEQ ID NO:7, the HVR-L2 comprises the amino acid        sequence of SEQ ID NO:11, and the HVR-L3 comprises the amino        acid sequence of SEQ ID NO:16. In another such embodiment, the        HVR-H1 comprises the amino acid sequence of SEQ ID NO:2, the        HVR-L1 comprises the amino acid sequence of SEQ ID NO:8, the        HVR-L2 comprises the amino acid sequence of SEQ ID NO:12, and        the HVR-L3 comprises the amino acid sequence of SEQ ID NO:17. In        another such embodiment, the HVR-H1 comprises the amino acid        sequence of SEQ ID NO:2, the HVR-L1 comprises the amino acid        sequence of SEQ ID NO:9, the HVR-L2 comprises the amino acid        sequence of SEQ ID NO:13, and the HVR-L3 comprises the amino        acid sequence of SEQ ID NO:18. In any of the above embodiments,        the antibody further comprises at least one framework selected        from a human VH Acceptor 2 framework and a human VL kappa        subgroup I consensus framework.

In another aspect, a monoclonal antibody that binds to Notch2 NRR isprovided, wherein the antibody comprises a heavy chain variable domainhaving at least 90% sequence identity to an amino acid sequence selectedfrom SEQ ID NO:20-21 and a light chain variable domain having at least90% sequence identity to an amino acid sequence selected from SEQ IDNO:22-25. In one embodiment, the antibody comprises a heavy chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:20 and a light chain variable domain having atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:22.In one such embodiment, the heavy chain variable domain comprises theamino acid sequence of SEQ ID NO:20, and the light chain variable domaincomprises the amino acid sequence of SEQ ID NO:22. In anotherembodiment, the antibody comprises a heavy chain variable domain havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:21 and a light chain variable domain having at least 90% sequenceidentity to an amino acid sequence selected from SEQ ID NOs:23-25. Inone such embodiment, the heavy chain variable domain comprises the aminoacid sequence of SEQ ID NO:21, and the light chain variable domaincomprises an amino acid sequence selected from SEQ ID NOs:23-25.

In another aspect, an isolated antibody is provided that binds to thesame epitope as an antibody selected from Antibody D, Antibody D-1,Antibody D-2, or Antibody D-3. In another aspect, an isolated antibodyis provided that binds to the LNR-A domain and the HD-C domain ofNotch2.

In another aspect, an anti-Notch2 NRR antibody is an antibody fragmentselected from a Fab, Fab′-SH, Fv, scFv, or (Fab′)₂ fragment. In anotheraspect, an anti-Notch2 NRR antibody is a humanized, chimeric, or humanantibody.’

Any of the above embodiments may be present singly or in combination.

In another aspect, a method of inhibiting Notch2 activity is provided,the method comprising exposing a cell that expresses Notch2 to anantibody as in any of the above embodiments. In another aspect, a methodof treating a disorder associated with increased expression or activityof Notch2 is provided, the method comprising administering to a subjectin need thereof an effective amount of an antibody as in any of theabove embodiments. In another aspect, a method of treating a B-cellmalignancy is provided, the method comprising administering to a subjectin need thereof an effective amount of an antibody as in any of theabove embodiments. In another aspect, a method of treating melanoma isprovided, the method comprising administering to a subject in needthereof an effective amount of an antibody as in any of the aboveembodiments.

In another aspect, a method of treating a disorder associated withincreased expression or activity of Notch1 is provided, the methodcomprising coadministering to a subject in need thereof an effectiveamount of an anti-Notch1 NRR antibody and a therapeutic agent selectedfrom dexamethasone and tamoxifen, wherein the therapeutic agent reducesaltered intestinal cell differentiation caused by the antibody. In onesuch embodiment, the disorder is a T-cell malignancy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the H1, H2, and H3 heavy chain hypervariable region (HVR)sequences of anti-Notch2 NRR monoclonal antibodies designated AntibodyD, Antibody D-1, Antibody D-2, and Antibody D-3, as described in ExampleB(1). Amino acid positions are numbered according to the Kabat numberingsystem as described below.

FIG. 2 shows the L1, L2, and L3 light chain HVR sequences of anti-Notch2NRR monoclonal antibodies designated Antibody D, Antibody D-1, AntibodyD-2, and Antibody D-3, as described in Example B(1). Amino acidpositions are numbered according to the Kabat numbering system asdescribed below.

FIG. 3 shows an alignment of the heavy chain variable region sequencesof Antibody D, Antibody D-1, Antibody D-2, and Antibody D-3. HVRs areenclosed in boxes, as described in Example B(1).

FIG. 4 shows an alignment of the light chain variable region sequencesof Antibody D, Antibody D-1, Antibody D-2, and Antibody D-3. HVRs areenclosed in boxes.

FIGS. 5A and 5B show exemplary acceptor human variable heavy (VH)consensus framework sequences for use in practicing the instantinvention. Sequence identifiers are as follows:

-   -   human VH subgroup I consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:32, 33, 34, 35).    -   human VH subgroup I consensus frameworks “B,” “C,” and “D” minus        extended hypervariable regions (SEQ ID NOs:36, 37, 34, 35; SEQ        ID NOs:36, 37, 38, 35; and SEQ ID NOs:36, 37, 39, 35).    -   human VH subgroup II consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:40, 41, 42, 35).    -   human VH subgroup II consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:43, 44, 42, 35;        SEQ ID NOs:43, 44, 45, 35; and SEQ ID NOs:43, 44, 46, and 35).    -   human VH subgroup III consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:47, 48, 49, 35).    -   human VH subgroup III consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:50, 51, 49, 35;        SEQ ID NOs:50, 51, 52, 35; and SEQ ID NOs:50, 51, 53, 35).    -   human VH acceptor framework “A” minus Kabat CDRs (SEQ ID NOs:54,        48, 55, 35).    -   human VH acceptor frameworks “B” and “C” minus extended        hypervariable regions (SEQ ID NOs:50, 51, 55, 35; and SEQ ID        NOs:50, 51, 56, 35).    -   human VH acceptor 2 framework “A” minus Kabat CDRs (SEQ ID        NOs:54, 48, 57, 35).    -   human VH acceptor 2 framework “B,” “C,” and “D” minus extended        hypervariable regions (SEQ ID NOs:50, 51, 57, 35; SEQ ID NOs:50,        51, 58, 35; and SEQ ID NOs:50, 51, 59, 35).

FIG. 6 shows exemplary acceptor human variable light (VL) consensusframework sequences for use in practicing the instant invention.Sequence identifiers are as follows:

-   -   human VL kappa subgroup I consensus framework (κv1): SEQ ID        NOs:60, 61, 62, 63    -   human VL kappa subgroup II consensus framework (κv2): SEQ ID        NOs:64, 65, 66, 63    -   human VL kappa subgroup III consensus framework (κv3): SEQ ID        NOs:67, 68, 69, 63    -   human VL kappa subgroup IV consensus framework (κv4): SEQ ID        NOs:70, 71, 72, 63

FIG. 7 shows framework sequences of huMAb4D5-8 light and heavy chains.Numbers in superscript/bold indicate amino acid positions according toKabat.

FIG. 8 shows framework sequences of huMAb4D5-8 light and heavy chainswith the indicated modifications. Numbers in superscript/bold indicateamino acid positions according to Kabat.

FIGS. 9A and 9B show that anti-NRR1 and anti-NRR2 antibodies bindspecifically to their cognate receptors, as described in Example B(1).(A) ELISA assay measuring binding of anti-NRR1 (left panel) andanti-NRR2 (right panel) to purified NRR protein fragments from each ofthe four human (h) and murine (m) Notch receptors. Binding, depicted asA₅₆₀ on the y-axis is graphed vs. a titration of anti-NRR1 or anti-NRR2.(B) FACS assay measuring binding of anti-NRR1 (panels 1-6) or anti-NRR2(panels 7-12) to untransfected K1-CHO cells (panels 1, 4, 7 and 10),K1-CHO cells stably transfected with N-myc-Notch1 (panels 2, 5, 8 ad 11)or K1-CHO cells stably transfected with N-myc-Notch2 (panels 3, 6, 9 and12). The N-myc-Notch transgenes were expressed under control of theinducible tet promoter; bottom row, control expression in the absence ofinduction with doxycycline (−Dox); top row, induced expression followingaddition of doxycycline (+Dox); note that the K1-CHO line endogenouslyexpresses Notch2, which is detected by anti-NRR2 in the presence andabsence of doxycycline (for example, compare panels 7 and 10).

FIGS. 10A-C show that anti-NRR1 and anti-NRR2 specifically inhibitsignaling from their targeted receptors, including receptors carryingactivating mutations, as described in Examples B(2) and B(3). (A)Co-culture assay measuring anti-NRR1 inhibition of Notch1 signaling.NIH-3T3 cells stably transfected with Jag1 were used to induce Notchsignaling in NIH-3T3 cells stably transfected with Notch1 (except for“−Jag1”, which used untransfected NIH-3T3 cells in place of theJag1-expressing cells). Notch signaling was measured using a Notchreporter gene (CSL-dependent promoter driving expression of fireflyluciferase) and is expressed relative to control gene expression(constitutive promoter driving expression of Renilla luciferase)normalized to the DAPT conditions (defined as a value of 1). +Jag1,standard co-culture assay; DMSO, DAPT vehicle alone; DAPT, 5 μM in DMSO;α-gD, isotype control antibody at 2000 ng/ml; α-NRR1, anti-NRR1 antibodyat the indicated concentrations; the last four assays included 80 ng/mlof either α-NRR1 or α-gD plus either purified Notch1 or Notch2 NRRprotein fragments, as indicated (+NRR1 or +NRR2). (B) Co-culture assaymeasuring anti-NRR2 inhibition of Notch2 signaling. NIH-3T3 cells stablytransfected with Jag1 were used to induce Notch signaling in U87MGcells, which express high levels of Notch2. The assay was performed asdescribed in (A). (C) Co-culture assay measuring anti-NRR1 inhibition ofNotch1 signaling from wild-type or mutant Notch1 receptors. The assaywas performed as in (A) except that the receptor-expressing cells weregenerated by transient transfection of plasmids expressing the indicatedNotch1 receptors. WT, wild-type Notch1; ΔPEST, Notch1 lacking the PESTdomain; L1594P, Notch1 carrying the indicated constitutively-activatingpoint mutation; E25, 625 ng/ml isotype control antibody; α-NRR1, 625ng/ml anti-NRR1 antibody; DMSO, GSI vehicle alone; DAPT, 5 μM in DMSO;CmpE, 1 μM compound E in DMSO.

FIGS. 11A-D show that anti-NRR1 and anti-NRR2 function asreceptor-specific inhibitors in vivo, as described in Example B(4).Balb/c mice were injected four times every four days with 5 mg/kg ofα-gD isotype control, α-NRR1 or α-NRR2, and cells were harvested fromthe thymus or spleen on day 13, one day after the fourth dose. (A)Thymus weight measurements. Thymus weights (in mg) are expressedrelative to total body weight (in g). Values represent the mean plusstandard deviation for three mice per group. (B) Thymus cell count. (C)CD4 and CD8 FACS to identify CD4⁺/CD8⁺ double-positive T cells. Numbersrepresent the percentage of thymic cells in the double-negative,single-positive and double-positive populations. Relative to the anti-gDcontrol (77.5%), anti-NRR1 dramatically reduced the percentage of cellsin the CD4⁺/CD8⁺ population (5.89%) whereas anti-NRR2 (80%) had nosignificant effect. (D) CD21 and CD23 FACS to identify marginal zone Bcells. Numbers represent the mean percentages ±standard deviation (fromthree animals) of cells within the MZB gate, which is boxed; p valuesare also noted; representative dot plots from one of the three animalsin each group are graphed. Relative to the anti-gD control (6.61%),anti-NRR2 nearly eliminated MZB cells (0.97%) whereas anti-NRR1 (6%) hadno significant effect.

In FIGS. 12A-D, a 2.2 Å structure of anti-NRR1 Fab/NRR1 co-crystalindicates that anti-NRR1 simultaneously contacts the LNR-A, LNR-B andHD-C domains, as described in Example B(5). (A) Table summarizing thebinding of α-NRR1 or α-NRR2 to Notch1-NRR or Notch2 NRR chimeric proteinfragments. The indicated NRR protein fragments (dark blue, Notch1sequences; light blue, Notch2 sequences) were expressed as secretedproteins fused to alkaline phosphatase to enable rapid measurements ofantibody binding in a plate-based assay. After using alkalinephosphatase activity to normalize for NRR expression and secretion,culture medium containing the indicated NRR chimeric proteins was addedto a 96-well plate that had been coated with α-NRR1, α-NRR2 or anisotype control antibody (used to assess background binding, not shown).Antibody binding was assessed by measuring alkaline phosphatase activitythat remained bound to the plate. Y, strong binding; N, no bindingobserved; W, weak binding observed. (B) Structure of human Notch NRR1.NRR1 is shown as a C-alpha cartoon. The three calcium ions in the LNRmotifs are shown as spheres. The position of the S2 cleavage site ismarked with an arrow. (C) Superposition of NRR1 on NRR2 (chain A frompdb code 2OO4) based on structurally conserved atoms. NRR1 (shaded) andNRR2 (white) are shown as C-alpha traces. (D) Open-book view of theinterface between NRR1 (left) and α-NRR1 Fab (right, border betweenheavy and light chains shown as a black dashed line). The extent towhich the solvent accessible surface area is buried by complex formationis indicated. Residues that are buried by at least 50% are labeled, withidentical NRR residues in Notch1 and Notch2 labeled in black font (seealso FIG. 18).

FIGS. 13A-C show that anti-NRR1 causes endothelial cell hypersprouting,as described in Example B(6). (A) In vitro endothelial cell sproutingassay. HUVECs were coated on Cytodex beads and co-cultured with skinfibroblasts. Cultures were either mock treated (control) or treated with1 μM of DBZ, 5 μg/ml of α-NRR1 or 5 μg/ml of α-Dll4. Bar=100 μm. (B)Sprout length measurements from the cultures in (A). (C) Mouse neonateretinal assay for endothelial cell sprouting and angiogenesis. Mouseneonates were injected at P1 and P3 with the indicated antibodies, andretinas were prepared at P5 to visualize perfusion of isolectin or theKi67 marker for proliferation. Panels I and II, Bar=1 mm. Panels III-VIrepresent enlargements of part of panels I and II, bar=0.2 mm.

FIGS. 14A-E show that selective antibody blocking of Notch1 signalingdisrupts tumor angiogenesis and inhibits tumor growth, as described inExample B(7). Graphs for three tumor xenograft models showing tumorvolume (mean+/−SEM) versus time following treatment with the indicatedantibodies: α-Ragweed (negative control), α-VEGF or α-NRR1. P values areshown for the α-Ragweed vs. α-NRR1 comparison. (A) Calu6 model. (B) HM7model. (C) HM7 model with dose titration of α-NRR1. (D) Endothelial cellstaining in representative tumor sections from the Calu6 model shown in(A). The antibodies used in the xenograft model are shown at the top.DAPI and α-CD31 were used to stain DNA and endothelial cells,respectively. The bottom row of panels shows the merged images. Bar=50μm. (E) Quantification of the CD31 staining shown in (D). Using ImageJto quantify the CD31 and DAPI staining in images similar to those shownin (D), relative CD31 staining (CD31 staining normalized to DAPIstaining) was graphed for each of the three antibody treatments relativeto the α-Ragweed control, which was set to a value of 1; data representthe mean+/−standard deviation over 8 image fields.

FIGS. 15A-C show that selective antibody blocking of Notch1 issufficient to alter cell fate in the small intestine, as described inExample B(8). (A) Total body weight change (mean+/−standard deviation)vs. time. Mice were treated as described for FIG. 11 with anti-gDisotype control, LTβR-Fc, anti-NRR1 or anti-NRR2. Arrows mark the daysof dosing. (B) Immunohistochemical analyses of small intestines. Micewere treated with the indicated concentrations of anti-gD isotypecontrol, DBZ or anti-NRR1 on days 0, 2 and 6, and small intestines wereprepared for immunohistochemical analyses on day 7. In each row asindicated, staining is shown for haemotoxylin and eosin (H & E), AlcianBlue for mucin and to mark secretory goblet cells, lysozyme to markPaneth cells, Ki-67 for proliferation and Hes1 as a Notch downstreamtarget. See FIGS. 19 and 20 for analyses of the large intestine at day 7and of the small intestine at day 2, respectively. Bar=40 μm. (C)Comparison of Notch1- versus Notch2-specific inhibition on smallintestinal cell differentiation. Small intestines from the studydescribed in (A) were prepared for Alcian Blue and Ki-67 one day afterthe final dosing with α-gD, α-NRR1 or α-NRR2, as indicated. Bar=50 μm.

FIG. 16 shows that anti-NRR1 is a potent inhibitor of Notch1 signalinginduced by multiple ligands, as described in Example B(2). Co-cultureassays of Notch1 signaling were performed as described in FIG. 10Aexcept that the ligand-expressing cells expressed Jag1, Jag2 or Dll1, asindicated.

FIG. 17 shows low-yield Notch1 NRR/Notch2 NRR chimeric proteinconstructs, as described in Example B(5). The table supplements FIG.12A, listing the NRR chimeric proteins that yielded little or nosecreted alkaline phosphatase activity, suggesting that the chimeraswere misfolded or otherwise unstable. For those fragments that yieldedweak but detectable alkaline phosphatase activity, the binding of α-NRR1or α-NRR2 is summarized. ---, no expression and binding not testable; N,no expression or binding; W, weak expression or binding.

FIG. 18 shows conservation of NRR1 residues contacted by anti-NRR1, asdescribed in Example B(1) and B(5). Amino acid sequence alignment of thehuman Notch1 NRR domain (SEQ ID NO:26), mouse Notch1 NRR domain (SEQ IDNO:27), human Notch2 NRR domain (SEQ ID NO:28) and mouse Notch2 NRRdomain (SEQ ID NO:29), with the sub-domain boundaries shown at theright. Human Notch1 NRR is listed as the reference sequence, andresidues in the other NRR sequences that are identical to those in humanNotch1 are shown in bold font. Residues that are at least 25% buried inthe anti-NRR1-Fab/Notch1-NRR structure (FIG. 12) are outlined, withsolid lines versus dashed lines reflecting an increased degree to whichthe residues are buried. Of the 21 amino acids that are at least 25%buried in the human Notch1 sequence, all 21 are identical in the mouseNotch1 sequence but only six are identical in the human and mouse Notch2sequences (plus a seventh conservative difference of T in place of S athuman Notch1 S1712); of these identical and “buried” residues in theNotch2 sequences, none are in the class of >75% buried. This sequencecomparison is consistent with (a) strong anti-NRR1 binding(approximately equal affinities) to both human and mouse Notch1 and (b)a lack of anti-NRR1 binding to human and mouse Notch2. Moreover, theburied residues all lay within the LNR-A, LNR-B and HD-C, consistentwith the domain swap experiment shown in FIGS. 11A and 17.

FIG. 19 shows that selective antibody blocking of Notch1 is sufficientto alter cell fate in the large intestine, as described in Example B(8).Histopathological analyses of large intestine samples taken at day 7from the experiment described in FIG. 15B.

FIG. 20 shows that intestinal cell fate changes develop 2 days afterblocking Notch1, as described in Example B(8). Histopathologicalanalyses of small intestine samples taken at day 2 from the experimentdescribed in FIG. 15B, which shows samples taken at day 7.

FIG. 21 shows that anti-NRR1 does not block Notch2-induced signaling invitro, as described in Example B(2). Co-culture assay in U87MG cells, asdescribed in FIG. 10B. Although anti-NRR1 potently inhibits Notch1signaling in vitro (FIG. 10A) and in vivo (FIGS. 11A-C), it does notaffect Notch2 signaling, even when used at a concentration (1000 ng/ml)that is over 100-fold above the IC₅₀. This result is consistent with thespecific binding of anti-NRR1 to Notch1 (FIGS. 9 and 12) as well as thelack of anti-NRR1 inhibition of Notch2 in vivo (FIG. 11D).

FIG. 22 shows possible synergistic effects of anti-NRR1 and anti-NRR2 onintestinal cell differentiation, as described in Example B8.

FIG. 23 shows that dexamethasone at least partially rescues theintestinal phenotype caused by anti-NRR1, as described in Example B(9).

FIGS. 24A and B show that selective blocking of either Notch1 or Notch2minimizes or avoids goblet cell metaplasia associated with pan-Notchinhibition, whereas blocking both Notch1 and Notch2 causes severe gobletcell metaplasia. (A) As described in Example B8, mice were dosed with 5mg/kg of anti-NRR1, anti-NRR2, or both, or a negative control antibodyanti-gD on the days marked with arrows; total body weight change(mean+/−standard deviation) vs. time is shown. (B) Immunohistochemicalanalyses of small intestines from mice treated as in (A), using AlcianBlue for mucin to mark secretory goblet cells.

FIGS. 25A and 25B show that anti-NRR2 (referred to as “anti-N2”)inhibits the growth of human melanoma cell lines SK23 and LOX-IMVI invitro, as described in Example B(10).

FIG. 26 shows the effect of anti-NRR2 (referred to as “anti-Notch2”) onfive diffuse large B-cell lymphoma (DLBCL) cell lines (listed on theright). As described in Example B(11), growth of one of the cell lines,“DB,” was strongly inhibited by treatment with anti-NRR2.

FIG. 27 shows the effect of anti-NRR2 (referred to as “anti-Notch2”) onthe the growth of the DB DLBCL cell line over time, as described inExample B(11).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The invention provides isolated antibodies that bind to Notch andmethods of using the same, e.g., for the diagnosis or treatment ofdiseases associated with expression or activity of Notch.

I. General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993).

II. Definitions

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with any document incorporatedherein by reference, the definition set forth below shall control.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “anti-Notch1 NRR antibody” or “an antibody that binds to Notch1NRR” refers to an antibody that is capable of binding Notch1 NRR withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting Notch1. Preferably, the extent ofbinding of an anti-Notch1 NRR antibody antibody to an unrelated,non-Notch protein is less than about 10% of the binding of the antibodyto Notch1 NRR as measured, e.g., by a radioimmunoassay (RIA). In certainembodiments, an antibody that binds to Notch1 NRR has a dissociationconstant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In certainembodiments, an anti-Notch1 NRR antibody binds to an epitope of Notchthat is conserved among Notch from different species, e.g., rodents(mice, rats) and primates.

The term “anti-Notch2 NRR antibody” or “an antibody that binds to Notch2NRR” refers to an antibody that is capable of binding Notch2 NRR withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting Notch2. Preferably, the extent ofbinding of an anti-Notch2 NRR antibody antibody to an unrelated,non-Notch protein is less than about 10% of the binding of the antibodyto Notch2 NRR as measured, e.g., by a radioimmunoassay (RIA). In certainembodiments, an antibody that binds to Notch2 NRR has a dissociationconstant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In certainembodiments, an anti-Notch2 NRR antibody binds to an epitope of Notchthat is conserved among Notch from different species, e.g., rodents(mice, rats) and primates.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co.,2000). An antibody may be part of a larger fusion molecule, formed bycovalent or non-covalent association of the antibody with one or moreother proteins or peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

A “naked antibody” for the purposes herein is an antibody that is notconjugated to a cytotoxic moiety or radiolabel.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo etal., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132(2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016;Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal., Nature Biotechnol. 14: 845-851 (1996); Neuberger, NatureBiotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include PRIMATIZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system(e.g., see U.S. Patent Application Publication No. US 2008/0181888 A1,Figures for EU numbering).

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies may be produced using certainprocedures known in the art. For example, Marks et al. Bio/Technology10:779-783 (1992) describes affinity maturation by VH and VL domainshuffling. Random mutagenesis of HVR and/or framework residues isdescribed by, for example, Barbas et al. Proc Nat. Acad. Sci. USA91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton etal. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol.154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896(1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “agonist antibody,” as used herein, is an antibody which partially orfully mimics at least one of the functional activities of a polypeptideof interest.

“Growth inhibitory” antibodies are those that prevent or reduceproliferation of a cell expressing an antigen to which the antibodybinds. For example, the antibody may prevent or reduce proliferation ofcancer cells in vitro and/or in vivo.

Antibodies that “induce apoptosis” are those that induce programmed celldeath as determined by standard apoptosis assays, such as binding ofannexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmicreticulum, cell fragmentation, and/or formation of membrane vesicles(called apoptotic bodies).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an antibody variable domain) and can be assessed usingvarious assays as disclosed, for example, in definitions herein.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see, e.g., Daëron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie etal., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol.Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

Binding to human FcRn in vivo and serum half life of human FcRn highaffinity binding polypeptides can be assayed, e.g., in transgenic miceor transfected human cell lines expressing human FcRn, or in primates towhich the polypeptides with a variant Fc region are administered. WO2000/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al. J. Biol.Chem. 9(2):6591-6604 (2001).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, andneutrophils. The effector cells may be isolated from a native source,e.g., from blood.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. NK cells, neutrophils, andmacrophages) enable these cytotoxic effector cells to bind specificallyto an antigen-bearing target cell and subsequently kill the target cellwith cytotoxins. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. No. 5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta), may be performed. Useful effector cells for suchassays include PBMC and NK cells. Alternatively, or additionally, ADCCactivity of the molecule of interest may be assessed in vivo, e.g., inan animal model such as that disclosed in Clynes et al. PNAS (USA)95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “Fc region-comprising antibody” refers to an antibody thatcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the antibody or by recombinant engineering of thenucleic acid encoding the antibody. Accordingly, a compositioncomprising an antibody having an Fc region according to this inventioncan comprise an antibody with K447, with all K447 removed, or a mixtureof antibodies with and without the K447 residue.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative and exemplary embodimentsfor measuring binding affinity are described in the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay. Solution binding affinity of Fabs for antigen ismeasured by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (see, e.g., Chen, et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% TWEEN-20™ inPBS. When the plates have dried, 150 μl/well of scintillant(MICROSCINT-20™; Packard) is added, and the plates are counted on aTOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations ofeach Fab that give less than or equal to 20% of maximal binding arechosen for use in competitive binding assays.

According to another embodiment, the Kd or Kd value is measured by usingsurface plasmon resonance assays using a BIACORE®-2000 or aBIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NETS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% TWEEN-20™ surfactant (PB ST) at 25° C. at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate,” “rate of association,” “association rate,” or “k_(on),”according to this invention can also be determined as described aboveusing a BIACORE®-2000 or a BIACORE®-3000 system (BIAcore, Inc.,Piscataway, N.J.).

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

An “acceptor human framework” or a “human acceptor framework” for thepurposes herein is a framework comprising the amino acid sequence of aVL or VH framework derived from a human immunoglobulin framework or ahuman consensus framework. An acceptor human framework “derived from” ahuman immunoglobulin framework or a human consensus framework maycomprise the same amino acid sequence thereof, or it may containpre-existing amino acid sequence changes. In some embodiments, thenumber of pre-existing amino acid changes are 10 or less, 9 or less, 8or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 orless. Where pre-existing amino acid changes are present in a VH,preferably those changes occur at only three, two, or one of positions71H, 73H and 78H; for instance, the amino acid residues at thosepositions may be 71A, 73T and/or 78A. In one embodiment, the VL acceptorhuman framework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., supra. In one embodiment, for the VL, the subgroup issubgroup kappa I as in Kabat et al., supra. In one embodiment, for theVH, the subgroup is subgroup III as in Kabat et al., supra.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, a human acceptor framework isderived from the VH subgroup III consensus framework and comprises anamino acid sequence comprising at least a portion or all of each of thefollowing sequences: (SEQ ID NO:50)-H1-(SEQ ID NO:51)-H2-(SEQ ID NO:57or 59)-H3-(SEQ ID NO: 35). In some embodiments, the last residue (S11)of SEQ ID NO:35 is substituted with an alanine.

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: (SEQ ID NO:60)-L1-(SEQ IDNO:61)-L2-(SEQ ID NO:62)-L3-(SEQ ID NO:63).

“Purified” means that a molecule is present in a sample at aconcentration of at least 95% by weight, or at least 98% by weight ofthe sample in which it is contained.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isseparated from at least one other nucleic acid molecule with which it isordinarily associated, for example, in its natural environment. Anisolated nucleic acid molecule further includes a nucleic acid moleculecontained in cells that ordinarily express the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO, or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle-stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

The term “Notch,” as used herein, refers to any native Notch (Notch1-4)from any vertebrate source, including mammals such as primates (e.g.humans) and rodents (e.g., mice and rats), unless otherwise indicated.The term encompasses “full-length,” unprocessed Notch as well as anyform of Notch that results from processing in the cell. The term alsoencompasses naturally occurring variants of Notch, e.g., splice variantsor allelic variants.

The term “Notch1,” as used herein, refers to any native Notch1 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed Notch1 as well as any form ofNotch1 that results from processing in the cell. The term alsoencompasses naturally occurring variants of Notch1, e.g., splicevariants or allelic variants.

The term “Notch2,” as used herein, refers to any native Notch2 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed Notch2 as well as any form ofNotch2 that results from processing in the cell. The term alsoencompasses naturally occurring variants of Notch2, e.g., splicevariants or allelic variants.

The term “Notch2 activity” refers to Notch2 signaling. An agent (e.g.,an antibody) that “inhibits Notch2 activity” significantly decreasesNotch2 signaling relative to the level of Notch2 signaling observed inan appropriate control under substantially identical conditions. Incertain embodiments, Notch2 activity may be assessed by a suitablereporter assay, as described herein in Example B(2). In certainembodiments, Notch2 activity may be assessed by measuring generation ofmarginal B zone cells, as described herein in Example B(4). In certainembodiments, the decrease in Notch2 signaling is at least 2-, 3-, 4-,5-, or 10-fold below the level observed in the control.

The term “Notch2 NRR” refers to a region of Notch2 consisting of thethree LNR modules (LNR-A, LNR-B, and LNR-C) and the HD domain (HD-N andHD-C). Exemplary human and mouse Notch2 NRR sequences are shown in FIG.18 (SEQ ID NOs:28 and 29, respectively). The Notch2 NRR may consist ofnon-covalently linked fragments, e.g., that result from the processingof Notch2 at S1, as well as a single contiguous polypeptide sequence. Byway of example and with reference to FIG. 18, human Notch2 NRR mayconsist of amino acids 1422-1677 of human Notch2 (SEQ ID NO:75), oralternatively, amino acids 1422-1608 of SEQ ID NO:75 noncovalentlylinked to amino acids 1609-1677 of SEQ ID NO:75.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

A “disorder” is any condition or disease that would benefit fromtreatment with a composition or method of the invention. This includeschronic and acute disorders including those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include conditions such ascancer.

“B-cell malignancies,” also called “B-cell neoplasms,” include but arenot limited to Hodgkin's disease (e.g., lymphocyte predominant Hodgkin'sdisease (LPHD)); non-Hodgkin's lymphoma (NHL); follicular center cell(FCC) lymphomas; acute lymphocytic leukemia (ALL, also called B-ALL);precursor B-lymphoblastic leukemia; chronic lymphocytic leukemia (CLL,also called B-CLL); Hairy cell leukemia; marginal zone B-cell lymphomas(including nodal, MALT, and splenic types); multiple myeloma(plasmacytoma, plasma cell myeloma); small non-cleaved cell lymphomas(e.g., Burkitt's lymphoma); large cell lymphomas (including diffuselarge cell (B-cell), diffuse mixed cell, and immunoblastic lymphoma);mantle cell lymphoma; small lymphocytic lymphoma; AIDS-related lymphomaand Waldenstrom's macroglobulinemia. Indolent lymphoma is aslow-growing, incurable disease in which the average patient survivesbetween six and 10 years following numerous periods of remission andrelapse; aggressive lymphomas are fast growing forms of lymphomaencompassing high grade and some categories of intermediate grade NHLs.

The term “melanoma” refers to a malignant tumor of melanocytes, whichare found primarily in the skin but which are also found in the eye(uveal melanoma) or in the intestines.

As used herein, “treatment” (and variations such as “treat” or“treating”) refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder or to slow the progression of adisease or disorder.

An “individual,” “subject,” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, farm animals (such as cows), sport animals, pets (such ascats, dogs, and horses), primates, mice and rats. In certainembodiments, a mammal is a human.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations may be sterile.

A “sterile” formulation is aseptic or free from all livingmicroorganisms and their spores.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, to elicit a desired response in the individual. Atherapeutically effective amount encompasses an amount in which anytoxic or detrimental effects of the substance/molecule are outweighed bythe therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,but not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount would be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. The term is intended to include radioactive isotopes (e.g.At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu), chemotherapeutic agents (e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, and the various antitumor or anticancer agents disclosed below.Other cytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

A “toxin” is any substance capable of having a detrimental effect on thegrowth or proliferation of a cell.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e. g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®,Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib),proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (seedefinition below); tyrosine kinase inhibitors (see definition below);serine-threonine kinase inhibitors such as rapamycin (sirolimus,RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636,SARASAR™); and pharmaceutically acceptable salts, acids or derivativesof any of the above; as well as combinations of two or more of the abovesuch as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone; andFOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens with mixed agonist/antagonist profile, including,tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®),idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, andselective estrogen receptor modulators (SERMs) such as SERM3; pureanti-estrogens without agonist properties, such as fulvestrant(FASLODEX®), and EM800 (such agents may block estrogen receptor (ER)dimerization, inhibit DNA binding, increase ER turnover, and/or suppressER levels); aromatase inhibitors, including steroidal aromataseinhibitors such as formestane and exemestane (AROMASIN®), andnonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®),letrozole (FEMARA®) and aminoglutethimide, and other aromataseinhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®),fadrozole, and 4(5)-imidazoles; lutenizing hormone-releasing hormoneagonists, including leuprolide (LUPRON® and ELIGARD®), goserelin,buserelin, and tripterelin; sex steroids, including progestines such asmegestrol acetate and medroxyprogesterone acetate, estrogens such asdiethylstilbestrol and premarin, and androgens/retinoids such asfluoxymesterone, all transretionic acid and fenretinide; onapristone;anti-progesterones; estrogen receptor down-regulators (ERDs);anti-androgens such as flutamide, nilutamide and bicalutamide; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell (such as a cell expressingNotch) either in vitro or in vivo. Thus, the growth inhibitory agent maybe one which significantly reduces the percentage of cells (such as acell expressing Notch) in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in Mendelsohn and Israel, eds., TheMolecular Basis of Cancer, Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W.B. Saunders,Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel)are anticancer drugs both derived from the yew tree. Docetaxel(TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is asemisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb).Paclitaxel and docetaxel promote the assembly of microtubules fromtubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

III. Compositions and Methods

The invention is based in part on the identification of Notch bindingagents, e.g., antibodies and fragments thereof. In specific embodiments,the invention concerns Notch2 negative regulatory region (NRR) bindingagents, such as isolated antibodies that bind Notch2 NRR and fragmentsthereof. Such antibodies are useful, e.g., for the diagnosis ortreatment of disorders associated with expression and/or activity ofNotch2 (e.g., increased expression or activity). In specificembodiments, anti-Notch2 NRR antibodies are useful for the diagnosis ortreatment of cancer, e.g., B-cell malignancies and melanoma.Accordingly, the invention provides methods, compositions, kits, andarticles of manufacture related to Notch2 NRR binding.

A. Anti-Notch2 NRR Antibodies

1. Exemplary Phage Library-Derived Antibodies

Exemplary monoclonal antibodies that were derived from a phage libraryare provided herein, as described in Example B. An antibody designated“Antibody D” that binds to Notch2 NRR was isolated. That antibody wasaffinity matured to generate antibodies designated Antibody D-1,Antibody D-2, and Antibody D-3. The sequences of the heavy chain andlight chain hypervariable regions (HVRs) of Antibody D, Antibody D-1,Antibody D-2, and Antibody D-3 are shown in FIGS. 1 and 2. The sequencesof the heavy and light chain variable domains of Antibody D, AntibodyD-1, Antibody D-2, and Antibody D-3 are shown in FIGS. 3 and 4. Furtherembodiments of anti-Notch2 NRR antibodies are provided as follows.

In one aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises at least one, two, three, four,five, or six HVRs selected from:

-   -   (a) an HVR-H1 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:3;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:10;    -   (e) an HVR-L2 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:14; and    -   (f) an HVR-L3 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:19.        In a further aspect, the antibody comprises an HVR-H3 comprising        the amino acid sequence of SEQ ID NO:5 and at least one, two,        three, four, or five HVRs selected from (a), (b), (d), (e),        and (f) above. In a further aspect, the antibody comprises (a),        (b), (c), (d), (e), and (f) above. With respect to (a), (d),        (e), and (f), any one or more of the following embodiments are        contemplated: HVR-H1 comprises an amino acid sequence selected        from SEQ ID NOs:1-2; HVR-L1 comprises an amino acid sequence        selected from SEQ ID NOs:6-9; HVR-L2 comprises an amino acid        sequence selected from SEQ ID NOs:11-13; and HVR-L3 comprises an        amino acid sequence selected from SEQ ID NOs:15-18.

In another aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises an HVR-H1 comprising an aminoacid sequence that conforms to the consensus sequence of SEQ ID NO:3, anHVR-H2 comprising the amino acid sequence of SEQ ID NO:4, and an HVR-H3comprising the amino acid sequence of SEQ ID NO:5. In one embodiment,HVR-H1 comprises an amino acid sequence selected from SEQ ID NOs:1-2.

In another aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises an HVR-L1 comprising an aminoacid sequence that conforms to the consensus sequence of SEQ ID NO:10,an HVR-L2 comprising an amino acid sequence that conforms to theconsensus sequence of SEQ ID NO:14, and an HVR-L3 comprising an aminoacid sequence that conforms to the consensus sequence of SEQ ID NO:19.The following embodiments are contemplated in any combination: HVR-L1comprises an amino acid sequence selected from SEQ ID NOs:6-9; HVR-L2comprises an amino acid sequence selected from SEQ ID NOs:11-13; andHVR-L3 comprises an amino acid sequence selected from SEQ ID NOs:15-18.In one embodiment, an antibody that binds to Notch2 NRR comprises anHVR-L1 comprising the amino acid sequence of SEQ ID NO:6; an HVR-L2comprising the amino acid sequence of SEQ ID NO:11; and an HVR-L3comprising the amino acid sequence of SEQ ID NO:15. In anotherembodiment, an antibody that binds to Notch2 NRR comprises an HVR-L1comprising the amino acid sequence of SEQ ID NO:7; an HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:11; and an HVR-L3 comprising theamino acid sequence of SEQ ID NO:16. In another embodiment, an antibodythat binds to Notch2 NRR comprises an HVR-L1 comprising the amino acidsequence of SEQ ID NO:8; an HVR-L2 comprising the amino acid sequence ofSEQ ID NO:12; and an HVR-L3 comprising the amino acid sequence of SEQ IDNO:17. In another embodiment, an antibody that binds to Notch2 NRRcomprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:9;an HVR-L2 comprising the amino acid sequence of SEQ ID NO:13; and anHVR-L3 comprising the amino acid sequence of SEQ ID NO:18.

In one embodiment, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:6;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:11; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:15.

In another embodiment, an antibody that specifically binds to Notch2 NRRis provided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:7;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:11; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:16.

In another embodiment, an antibody that specifically binds to Notch2 NRRis provided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:8;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:12; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:17.

In another embodiment, an antibody that specifically binds to Notch2 NRRis provided, wherein the antibody comprises:

-   -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:2;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5;    -   (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:9;    -   (e) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:13; and    -   (f) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:18.

In certain embodiments, any of the above antibodies further comprises atleast one framework selected from a VH subgroup III consensus frameworkand a VL subgroup I consensus framework.

In certain embodiments, an anti-Notch2 NRR antibody is affinity matured.For example, any one or more of the following substitutions in theindicated HVR positions (Kabat numbered) may be made in any combination:

-   -   in HVR-H1 (SEQ ID NO:1): S28T; T305;    -   in HVR-L1 (SEQ ID NO:6): S28N; I29N or V; S30R or K; S31R; Y32F    -   in HVR-L2 (SEQ ID NO:11): G50R; S53I or T; A55E    -   in HVR-L3 (SEQ ID NO:15): S93I or R; L96W or H        The specific antibodies disclosed herein, i.e., Antibody D as        well as affinity matured forms of Antibody D (D-1, D-2, and        D-3), may undergo further affinity maturation. Accordingly,        affinity matured forms of any of the antibodies described herein        are provided.

In certain embodiments, an anti-Notch2 NRR antibody having any of theabove HVR sequences can further comprise any suitable framework variabledomain sequence, provided binding activity to Notch2 NRR issubstantially retained. In certain embodiments, an anti-Notch2 NRRantibody comprises a human variable heavy (VH) consensus frameworksequence, as in any of the VH consensus framework sequences shown inFIGS. 5A and 5B. In one embodiment, the VH consensus framework sequencecomprises a human subgroup III heavy chain framework consensus sequence,e.g., as shown in FIGS. 5A and 5B. In another embodiment, the VHconsensus framework sequence comprises an “Acceptor 2” frameworksequence, e.g., as shown in FIGS. 5A and 5B. In a particular embodiment,the VH framework consensus sequence comprises FR1-FR4 of Acceptor 2B orAcceptor 2D, wherein the FR4 comprises SEQ ID NO:35 (FIGS. 5A and 5B),with the last residue of SEQ ID NO:35 (S11) optionally being substitutedwith alanine. In a further particular embodiment, the VH frameworkconsensus sequence comprises the sequences of SEQ ID NOs:50; 51; 57 or59; and 35, wherein S11 of SEQ ID NO:35 is optionally substituted withalanine.

In certain embodiments, an anti-Notch2 NRR antibody having any of theabove HVR sequences can further comprise a human variable light (VL)consensus framework sequence as shown in FIGS. 6A and 6B. In oneembodiment, the VL consensus framework sequence comprises a human VLkappa subgroup I consensus framework (κv1) sequence, e.g., as shown inFIGS. 6A and 6B. In another embodiment, the VL framework consensussequence comprises FR1-FR4 of huMAb4D5-8 as shown in FIG. 7 or 8. In aparticular embodiment, the VL framework consensus sequence comprises thesequences of SEQ ID NOs:60, 61, 62, and 63.

In another aspect, an anti-Notch2 NRR antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs:20-21. In certain embodiments, a VHsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-Notch2 NRR antibody comprising that sequence retains the ability tobind to Notch2 NRR. In certain embodiments, a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in an amino acidsequence selected from SEQ ID NOs:20-21. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). In a particular embodiment, the VH comprisesone, two or three HVRs selected from: (a) an HVR-H1 comprising an aminoacid sequence that conforms to the consensus sequence of SEQ ID NO:3,(b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c)an HVR-H3 comprising the amino acid sequence of SEQ ID NO:5. In one suchembodiment, HVR-H1 comprises an amino acid sequence selected from SEQ IDNOs:1-2.

In another aspect, an antibody that specifically binds to Notch2 NRR isprovided, wherein the antibody comprises a light chain variable domain(VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to an amino acid sequence selected from SEQ IDNOs:22-25. In certain embodiments, a VL sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-Notch2 NRRantibody comprising that sequence retains the ability to bind to Notch2NRR. In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in an amino acid sequence selectedfrom SEQ ID NOs:22-25. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). In a particular embodiment, the VL comprises one, two or threeHVRs selected from (a) an HVR-L1 comprising an amino acid sequence thatconforms to the consensus sequence of SEQ ID NO:10; (b) an HVR-L2comprising an amino acid sequence that conforms to the consensussequence of SEQ ID NO:14; and (c) an HVR-L3 comprising an amino acidsequence that conforms to the consensus sequence of SEQ ID NO:19. In onesuch embodiment, the VL comprises one, two or three HVRs selected from(a) an HVR-L1 comprising an amino acid sequence selected from SEQ IDNOs:6-9; (b) an HVR-L2 comprising an amino acid sequence selected fromSEQ ID NOs:11-13; and (c) an HVR-L3 comprising an amino acid sequenceselected from SEQ ID NOs:15-18. In one such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:6; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:11; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:15. In another such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:7; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:11; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:16. In another such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:8; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:12; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:17. In another such embodiment, the VL comprisesone, two or three HVRs selected from (a) an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:9; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:13; and (c) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:18.

In certain embodiments of the variant VH and VL sequences providedabove, substitutions, insertions, or deletions may occur within theHVRs. In such embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations that do not substantially reducebinding affinity may be made in HVRs. In certain instances, alterationsin HVRs may actually improve antibody affinity. Such alterations may bemade in HVR “hotspots” (i.e., residues encoded by codons that undergomutation at high frequency during the somatic maturation process) inorder to increase antibody affinity. (See, e.g., Chowdhury, Methods Mol.Biol. 207:179-196, 2008.) In certain embodiments of the variant VH andVL sequences provided above, each HVR either is conserved (unaltered),or contains no more than a single amino acid substitution, insertion ordeletion.

In another aspect, an antibody that specifically binds Notch2 NRR isprovided, wherein the antibody comprises a VH as in any of theembodiments provided above, and a VL as in any of the embodimentsprovided above. In one embodiment, the antibody comprises a VH having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:20, and a VL having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:22. In one suchembodiment, the VH comprises one, two or three HVRs selected from: (a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) anHVR-H3 comprising the amino acid sequence of SEQ ID NO:5, and the VLcomprises one, two or three HVRs selected from (a) an HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:6; (b) an HVR-L2 comprising theamino acid sequence of SEQ ID NO:11; and (c) an HVR-L3 comprising theamino acid sequence of SEQ ID NO:15. In a particular embodiment, theantibody comprises a VH comprising the amino acid sequence of SEQ IDNO:20, and a VL comprising the amino acid sequence of SEQ ID NO:22.

In another embodiment, an anti-Notch2 NRR antibody that specificallybinds Notch2 NRR comprises a VH having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:21, and a VL having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID NOs:23-25. In one such embodiment, the VHcomprises one, two or three HVRs selected from: (a) an HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:2, (b) an HVR-H2 comprising theamino acid sequence of SEQ ID NO:4, and (c) an HVR-H3 comprising theamino acid sequence of SEQ ID NO:5, and the VL comprises one, two orthree HVRs selected from (a) an HVR-L1 comprising an amino acid sequenceselected from SEQ ID NOs:7-9; (b) an HVR-L2 comprising an amino acidsequence selected from SEQ ID NOs:11-13; and (c) an HVR-L3 comprising anamino acid sequence selected from SEQ ID NOs:16-18. In particularembodiments, the antibody comprises a VH comprising the amino acidsequence of SEQ ID NO:21 and a VL comprising an amino acid sequenceselected from SEQ ID NOs:23-25.

In certain embodiments, an affinity-matured form of any of the aboveantibodies is provided. In further embodiments, a recombinant proteinthat specifically binds Notch2 NRR is is provided, wherein therecombinant protein comprises an antigen binding site(s) of any of theabove antibodies. In one such embodiment, a recombinant proteincomprises any one or more of the HVRs provided above.

In certain embodiments, a polynucleotide encoding any of the aboveantibodies is provided. In one embodiment, a vector comprising thepolynucleotide is provided. In one embodiment, a host cell comprisingthe vector is provided. In one embodiment, the host cell is eukaryotic.In one embodiment, the host cell is a CHO cell. In one embodiment, amethod of making an anti-Notch2 NRR antibody is provided, wherein themethod comprises culturing the host cell under conditions suitable forexpression of the polynucleotide encoding the antibody, and isolatingthe antibody.

2. Further Exemplary Antibodies

In one embodiment, the invention provides an isolated anti-Notch2 NRRantibody that inhibits Notch2 activity. For example, the antibodies ofthe invention may modulate one or more aspects of Notch2 signaling,including disruption of any biologically relevant Notch2 signalingpathway.

In a further embodiment, the invention provides an isolated anti-Notch2NRR antibody that binds Notch2 NRR with a Kd of ≦100 nM. In certainembodiments, the anti-Notch2 NRR antibody binds the Notch2 NRR with a Kdof ≦10 nM, ≦1 nM, or ≦0.1 nM. As described in the Examples herein, theexemplary phage Antibody D-3 binds with a Kd of 5 nM. As iswell-established in the art, binding affinity of a ligand to itsreceptor can be determined using any of a variety of assays, andexpressed in terms of a variety of quantitative values. Accordingly, inone embodiment, the binding affinity is expressed as Kd values andreflects intrinsic binding affinity (e.g., with minimized avidityeffects). Generally and preferably, binding affinity is measured invitro, whether in a cell-free or cell-associated setting. Any of anumber of assays known in the art, including those described herein, canbe used to obtain binding affinity measurements, including, for example,Biacore, radioimmunoassay (RIA), and ELISA.

In another embodiment, an isolated antibody that binds to Notch2 NRR isprovided, wherein the antibody does not significantly bind to a Notchfamily member other than Notch2 (i.e., Notch1, 3, and 4 in mammals).Such an antibody may be identified using the assays provided in ExampleB(1). In one embodiment, the antibody binds to human Notch2 NRR and aNotch2 NRR from at least one other non-human species, e.g., mouse.

In another embodiment, an isolated antibody is provided that binds tothe same epitope as an antibody provided herein. In one embodiment, anisolated anti-Notch2 NRR antibody is provided that binds to the sameepitope as an antibody selected from Antibody D, Antibody D-1, AntibodyD-2, and Antibody D-3. In another embodiment, the invention provides ananti-Notch2 NRR antibody that competes with an antibody selected fromAntibody D, Antibody D-1, Antibody D-2, and Antibody D-3. In anotherembodiment, an isolated antibody is provided that binds to at least onedomain selected from the LNR-A domain and the HD-C domain of Notch2. Inone such embodiment, the antibody binds to both the LNR-A domain and theHD-C domain. In another such embodiment, the antibody further binds tothe LNR-B and/or HD-N domains. Those domains are delineated in FIG. 18.

The above embodiments of anti-Notch2 NRR antibodies may be presentsingly or in any combination. Furthermore, any anti-Notch2 NRR antibodydescribed herein may possess any one or more of the following features:In certain embodiments, an anti-Notch2 NRR antibody is a monoclonalantibody. In certain embodiments, an anti-Notch2 NRR antibody is anantibody fragment selected from a Fab, Fab′-SH, Fv, scFv, or (Fab′)₂fragment. In another embodiment, an anti-Notch2 NRR antibody is ahumanized, human or chimeric antibody.

3. Antibody Fragments

The present invention encompasses antibody fragments. Antibody fragmentsmay be generated by traditional means, such as enzymatic digestion, orby recombinant techniques. In certain circumstances there are advantagesof using antibody fragments, rather than whole antibodies. The smallersize of the fragments allows for rapid clearance, and may lead toimproved access to solid tumors. For a review of certain antibodyfragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

4. Humanized Antibodies

The invention encompasses humanized antibodies. Various methods forhumanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody. See, e.g., Sims et al.(1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol.196:901. Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. See, e.g., Carter et al. (1992) Proc.Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol.,151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

5. Human Antibodies

Human antibodies of the invention can be constructed by combining Fvclone variable domain sequence(s) selected from human-derived phagedisplay libraries with known human constant domain sequences(s) asdescribed above. Alternatively, human monoclonal antibodies of theinvention can be made by the hybridoma method. Human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

6. Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different antigens. In certainembodiments, bispecific antibodies are human or humanized antibodies. Incertain embodiments, one of the binding specificities is for Notch2 andthe other is for any other antigen. In certain embodiments, bispecificantibodies may bind to two different epitopes of Notch2. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress Notch2. These antibodies possess a Notch2-binding arm and an armwhich binds a cytotoxic agent, such as, e.g., saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion, forexample, is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In certainembodiments, the first heavy-chain constant region (CH1), containing thesite necessary for light chain binding, is present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The interfacecomprises at least a part of the C_(H)3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking method. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

7. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. In certain embodiments, the dimerization domain comprises(or consists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. In certain embodiments, amultivalent antibody comprises (or consists of) three to about eightantigen binding sites. In one such embodiment, a multivalent antibodycomprises (or consists of) four antigen binding sites. The multivalentantibody comprises at least one polypeptide chain (for example, twopolypeptide chains), wherein the polypeptide chain(s) comprise two ormore variable domains. For instance, the polypeptide chain(s) maycomprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein may further comprise atleast two (for example, four) light chain variable domain polypeptides.The multivalent antibody herein may, for instance, comprise from abouttwo to about eight light chain variable domain polypeptides. The lightchain variable domain polypeptides contemplated here comprise a lightchain variable domain and, optionally, further comprise a CL domain.

8. Single-Domain Antibodies

In some embodiments, an antibody of the invention is a single-domainantibody. A single-domain antibody is a single polypeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).In one embodiment, a single-domain antibody consists of all or a portionof the heavy chain variable domain of an antibody.

9. Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (e.g.,alanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody of the invention is altered toincrease or decrease the extent to which the antibody is glycosylated.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of a carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites) is created or removed. The alteration may also bemade by the addition, deletion, or substitution of one or more serine orthreonine residues to the sequence of the original antibody (forO-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. (1997) TIBTECH 15:26-32. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

For example, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. Such variants may have improved ADCC function. See, e.g., USPatent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which further improve ADCC, forexample, substitutions at positions 298, 333, and/or 334 of the Fcregion (Eu numbering of residues). Such substitutions may occur incombination with any of the variations described above.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for many applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the antibody are measured to ensurethat only the desired properties are maintained. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the antibody lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.The primary cells for mediating ADCC, NK cells, express Fc(RIII only,whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-92 (1991). Non-limiting examples of invitro assays to assess ADCC activity of a molecule of interest isdescribed in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I., et al.Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al.,Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, for example, Petkova, S. B. et al., Int'l. Immunol.18(12):1759-1769 (2006)).

Other antibody variants having one or more amino acid substitutions areprovided. Sites of interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions.” More substantial changes, denominated“exemplary substitutions” are provided in Table 1, or as furtherdescribed below in reference to amino acid classes. Amino acidsubstitutions may be introduced into an antibody of interest and theproducts screened, e.g., for a desired activity, such as improvedantigen binding, decreased immunogenicity, improved ADCC or CDC, etc.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Modifications in the biological properties of an antibody may beaccomplished by selecting substitutions that affect (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Amino acids may be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)):

-   -   (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe        (F), Trp (W), Met (M)    -   (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr        (Y), Asn (N), Gln (Q)    -   (3) acidic: Asp (D), Glu (E)    -   (4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, into theremaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have modified (e.g., improved) biologicalproperties relative to the parent antibody from which they aregenerated. An exemplary substitutional variant is an affinity maturedantibody, which may be conveniently generated using phage display-basedaffinity maturation techniques. Briefly, several hypervariable regionsites (e.g. 6-7 sites) are mutated to generate all possible amino acidsubstitutions at each site. The antibodies thus generated are displayedfrom filamentous phage particles as fusions to at least part of a phagecoat protein (e.g., the gene III product of M13) packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g. binding affinity). In order to identifycandidate hypervariable region sites for modification, scanningmutagenesis (e.g., alanine scanning) can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according totechniques known in the art, including those elaborated herein. Oncesuch variants are generated, the panel of variants is subjected toscreening using techniques known in the art, including those describedherein, and variants with superior properties in one or more relevantassays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of antibodies of the invention, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody of the invention maycomprise one or more alterations as compared to the wild typecounterpart antibody, e.g. in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter, Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants. WO00/42072(Presta) and WO 2004/056312 (Lowman) describe antibody variants withimproved or diminished binding to FcRs. The content of these patentpublications are specifically incorporated herein by reference. See,also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodieswith increased half lives and improved binding to the neonatal Fcreceptor (FcRn), which is responsible for the transfer of maternal IgGsto the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton etal.). These antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1, WO99/51642. The contents of those patent publicationsare specifically incorporated herein by reference. See, also, Idusogieet al. J. Immunol. 164: 4178-4184 (2000).

In another aspect, the invention provides antibodies comprisingmodifications in the interface of Fc polypeptides comprising the Fcregion, wherein the modifications facilitate and/or promoteheterodimerization. These modifications comprise introduction of aprotuberance into a first Fc polypeptide and a cavity into a second Fcpolypeptide, wherein the protuberance is positionable in the cavity soas to promote complexing of the first and second Fc polypeptides.Methods of generating antibodies with these modifications are known inthe art, e.g., as described in U.S. Pat. No. 5,731,168.

In yet another aspect, it may be desirable to create cysteine engineeredantibodies, e.g., “thioMAbs,” in which one or more residues of anantibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, as described further herein. Incertain embodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of theheavy chain Fc region.

10. Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Certain Methods of Making Antibodies

1. Certain Hybridoma-Based Methods

Monoclonal antibodies of the invention can be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), andfurther described, e.g., in Hongo et al., Hybridoma, 14 (3): 253-260(1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) regardinghuman-human hybridomas. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 regarding production of monoclonalhuman natural IgM antibodies from hybridoma cell lines. Human hybridomatechnology (Trioma technology) is described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

For various other hybridoma techniques, see, e.g., US 2006/258841; US2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507.An exemplary protocol for producing monoclonal antibodies using thehybridoma method is described as follows. In one embodiment, a mouse orother appropriate host animal, such as a hamster, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization. Antibodiesare raised in animals by multiple subcutaneous (sc) or intraperitoneal(ip) injections of a polypeptide comprising Notch2 or a fragment thereof(e.g., Notch2 NRR), and an adjuvant, such as monophosphoryl lipid A(MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc.,Hamilton, Mont.). A polypeptide comprising Notch2 or a fragment thereofmay be prepared using methods well known in the art, such as recombinantmethods, some of which are further described herein. Serum fromimmunized animals is assayed for anti-Notch2 antibodies, and boosterimmunizations are optionally administered. Lymphocytes from animalsproducing anti-Notch2 antibodies are isolated. Alternatively,lymphocytes may be immunized in vitro.

Lymphocytes are then fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. See, e.g.,Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986). Myeloma cells may be used that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells. Preferably, serum-free hybridoma cell culturemethods are used to reduce use of animal-derived serum such as fetalbovine serum, as described, for example, in Even et al., Trends inBiotechnology, 24(3), 105-108 (2006).

Oligopeptides as tools for improving productivity of hybridoma cellcultures are described in Franek, Trends in Monoclonal AntibodyResearch, 111-122 (2005). Specifically, standard culture media areenriched with certain amino acids (alanine, serine, asparagine,proline), or with protein hydrolyzate fractions, and apoptosis may besignificantly suppressed by synthetic oligopeptides, constituted ofthree to six amino acid residues. The peptides are present at millimolaror higher concentrations.

Culture medium in which hybridoma cells are growing may be assayed forproduction of monoclonal antibodies that bind to Notch2. The bindingspecificity of monoclonal antibodies produced by hybridoma cells may bedetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay(ELISA). The binding affinity of the monoclonal antibody can bedetermined, for example, by Scatchard analysis. See, e.g., Munson etal., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.See, e.g., Goding, supra. Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, hybridomacells may be grown in vivo as ascites tumors in an animal. Monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography. One procedure for isolation of proteins fromhybridoma cells is described in US 2005/176122 and U.S. Pat. No.6,919,436. The method includes using minimal salts, such as lyotropicsalts, in the binding process and preferably also using small amounts oforganic solvents in the elution process.

2. Certain Library Screening Methods

Antibodies of the invention can be made by using combinatorial librariesto screen for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are describedgenerally in Hoogenboom et al. in Methods in Molecular Biology 178:1-37(O'Brien et al., ed., Human Press, Totowa, N.J., 2001). For example, onemethod of generating antibodies of interest is through the use of aphage antibody library as described in Lee et al., J. Mol. Biol. (2004),340(5):1073-93.

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the antibodies of the invention can beobtained by designing a suitable antigen screening procedure to selectfor the phage clone of interest followed by construction of a fulllength antibody clone using the Fv sequences from the phage clone ofinterest and suitable constant region (Fc) sequences described in Kabatet al., Sequences of Proteins of Immunological Interest, Fifth Edition,NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody isformed from two variable (V) regions of about 110 amino acids, one eachfrom the light (VL) and heavy (VH) chains, that both present threehypervariable loops (HVRs) or complementarity-determining regions(CDRs). Variable domains can be displayed functionally on phage, eitheras single-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Asused herein, scFv encoding phage clones and Fab encoding phage clonesare collectively referred to as “Fv phage clones” or “Fv clones.”

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibodyfragments by fusion to the minor coat protein pIII. The antibodyfragments can be displayed as single chain Fv fragments, in which VH andVL domains are connected on the same polypeptide chain by a flexiblepolypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol.,222: 581-597 (1991), or as Fab fragments, in which one chain is fused topIII and the other is secreted into the bacterial host cell periplasmwhere assembly of a Fab-coat protein structure which becomes displayedon the phage surface by displacing some of the wild type coat proteins,e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-Notch2 clones (e.g., anti-Notch2 NRR clones) isdesired, the subject is immunized with Notch2 (or Notch2 NRR) togenerate an antibody response, and spleen cells and/or circulating Bcells other peripheral blood lymphocytes (PBLs) are recovered forlibrary construction. In a preferred embodiment, a human antibody genefragment library biased in favor of anti-Notch2 clones is obtained bygenerating an anti-Notch2 antibody response in transgenic mice carryinga functional human immunoglobulin gene array (and lacking a functionalendogenous antibody production system) such that Notch2 immunizationgives rise to B cells producing human antibodies against Notch2. Thegeneration of human antibody-producing transgenic mice is describedbelow.

Additional enrichment for anti-Notch2 reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing Notch2-specific membrane bound antibody, e.g., by cellseparation using Notch2 affinity chromatography or adsorption of cellsto fluorochrome-labeled Notch2 followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which Notch2 isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, library diversity is maximized by usingPCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities of about 10⁻⁹ M or less.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, Notch2 (or Notch2 NRR) can be used tocoat the wells of adsorption plates, expressed on host cells affixed toadsorption plates or used in cell sorting, or conjugated to biotin forcapture with streptavidin-coated beads, or used in any other method forpanning phage display libraries.

The phage library samples are contacted with immobilized Notch2 underconditions suitable for binding at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by Notch2 antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for Notch2.However, random mutation of a selected antibody (e.g. as performed insome affinity maturation techniques) is likely to give rise to manymutants, most binding to antigen, and a few with higher affinity. Withlimiting Notch2, rare high affinity phage could be competed out. Toretain all higher affinity mutants, phages can be incubated with excessbiotinylated Notch2, but with the biotinylated Notch2 at a concentrationof lower molarity than the target molar affinity constant for Notch2.The high affinity-binding phages can then be captured bystreptavidin-coated paramagnetic beads. Such “equilibrium capture”allows the antibodies to be selected according to their affinities ofbinding, with sensitivity that permits isolation of mutant clones withas little as two-fold higher affinity from a great excess of phages withlower affinity. Conditions used in washing phages bound to a solid phasecan also be manipulated to discriminate on the basis of dissociationkinetics.

Anti-Notch2 clones may be selected based on activity. In certainembodiments, the invention provides anti-Notch2 antibodies that bind toliving cells that naturally express Notch2. In one embodiment, theinvention provides anti-Notch2 antibodies that block the binding betweena Notch2 ligand and Notch2, but do not block the binding between aNotch2 ligand and a second protein. Fv clones corresponding to suchanti-Notch2 antibodies can be selected by (1) isolating anti-Notch2clones from a phage library as described above, and optionallyamplifying the isolated population of phage clones by growing up thepopulation in a suitable bacterial host; (2) selecting Notch2 and asecond protein against which blocking and non-blocking activity,respectively, is desired; (3) adsorbing the anti-Notch2 phage clones toimmobilized Notch2; (4) using an excess of the second protein to eluteany undesired clones that recognize Notch2-binding determinants whichoverlap or are shared with the binding determinants of the secondprotein; and (5) eluting the clones which remain adsorbed following step(4). Optionally, clones with the desired blocking/non-blockingproperties can be further enriched by repeating the selection proceduresdescribed herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones of the invention is readily isolated and sequenced usingconventional procedures (e.g. by using oligonucleotide primers designedto specifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

DNA encoding anti-Notch2 antibody derived from a hybridoma of theinvention can also be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofhomologous murine sequences derived from the hybridoma clone (e.g. as inthe method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855(1984)). DNA encoding a hybridoma- or Fv clone-derived antibody orfragment can be further modified by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of the Fvclone or hybridoma clone-derived antibodies of the invention.

3. Vectors, Host Cells, and Recombinant Methods

Antibodies may also be produced using recombinant methods. Forrecombinant production of an anti-Notch2 antibody, nucleic acid encodingthe antibody is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

a) Signal Sequence Component

An antibody of the invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process a native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,α factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

b) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

c) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upantibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS),thymidine kinase, metallothionein-I and -II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR gene are identified byculturing the transformants in a culture medium containing methotrexate(Mtx), a competitive antagonist of DHFR. Under these conditions, theDHFR gene is amplified along with any other co-transformed nucleic acid.A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFRactivity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified byculturing the transformants in a culture medium containing L-methioninesulfoximine (Msx), an inhibitor of GS. Under these conditions, the GSgene is amplified along with any other co-transformed nucleic acid. TheGS selection/amplification system may be used in combination with theDHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody of interest, wild-type DHFR gene, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

d) Promoter Component

Expression and cloning vectors generally contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding an antibody. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, β-lactamase and lactose promoter systems,alkaline phosphatase promoter, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding an antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

e) Enhancer Element Component

Transcription of a DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

f) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

g) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fusion proteins, and antibody fragmentscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) that by itself showseffectiveness in tumor cell destruction. Full length antibodies havegreater half life in circulation. Production in E. coli is faster andmore cost efficient. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et.al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523(Simmons et al.), which describes translation initiation region (TIR)and signal sequences for optimizing expression and secretion. See alsoCharlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli. After expression, the antibody may beisolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out similar to the processfor purifying antibody expressed e.g, in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger. For a reviewdiscussing the use of yeasts and filamentous fungi for the production oftherapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414(2004).

Certain fungi and yeast strains may be selected in which glycosylationpathways have been “humanized,” resulting in the production of anantibody with a partially or fully human glycosylation pattern. See,e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describinghumanization of the glycosylation pathway in Pichia pastoris); andGerngross et al., supra.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,duckweed (Lemnaceae), alfalfa (M. truncatula), and tobacco can also beutilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498,6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technologyfor producing antibodies in transgenic plants).

Vertebrate cells may be used as hosts, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure.Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Other useful mammalian host cell lines include Chinese hamsterovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl.Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NSO andSp2/0. For a review of certain mammalian host cell lines suitable forantibody production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003),pp. 255-268.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

h) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

i) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being among one of thetypically preferred purification steps. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

C. Immunoconjugates

The invention also provides immunoconjugates (interchangeably referredto as “antibody-drug conjugates,” or “ADCs”) comprising an antibodyconjugated to one or more cytotoxic agents, such as a chemotherapeuticagent, a drug, a growth inhibitory agent, a toxin (e.g., a proteintoxin, an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Immunoconjugates have been used for the local delivery of cytotoxicagents, i.e., drugs that kill or inhibit the growth or proliferation ofcells, in the treatment of cancer (Lambert, J. (2005) Curr. Opinion inPharmacology 5:543-549; Wu et al (2005) Nature Biotechnology23(9):1137-1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos(1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer(1997) Adv. Drug Deliv. Rev. 26:151-172; U.S. Pat. No. 4,975,278).Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and intracellular accumulation therein, where systemicadministration of unconjugated drugs may result in unacceptable levelsof toxicity to normal cells as well as the tumor cells sought to beeliminated (Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe(1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological And ClinicalApplications (A. Pinchera et al., eds) pp. 475-506. Both polyclonalantibodies and monoclonal antibodies have been reported as useful inthese strategies (Rowland et al., (1986) Cancer Immunol. Immunother.21:183-87). Drugs used in these methods include daunomycin, doxorubicin,methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins usedin antibody-toxin conjugates include bacterial toxins such as diphtheriatoxin, plant toxins such as ricin, small molecule toxins such asgeldanamycin (Mandler et al (2000) J. Nat. Cancer Inst.92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791),maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928;Hinman et al (1993) Cancer Res. 53:3336-3342). The toxins may exerttheir cytotoxic effects by mechanisms including tubulin binding, DNAbinding, or topoisomerase inhibition. Some cytotoxic drugs tend to beinactive or less active when conjugated to large antibodies or proteinreceptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and 111In or 90Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody-drug conjugate composed of a huCD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody-drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andother cancers. MLN-2704 (Millennium Pharm., BZL Biologics, ImmunogenInc.), an antibody-drug conjugate composed of the anti-prostate specificmembrane antigen (PSMA) monoclonal antibody linked to the maytansinoiddrug moiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnol. 21(7):778-784)and are under therapeutic development.

In certain embodiments, an immunoconjugate comprises an antibody and achemotherapeutic agent or other toxin. Chemotherapeutic agents useful inthe generation of immunoconjugates are described herein (e.g., above).Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct.28, 1993. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

1. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×105 HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. Patent ApplicationPublication No. US 2005/016993 A1, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed in US2005/016993 A1. The linking groups include disulfide groups, thioethergroups, acid labile groups, photolabile groups, peptidase labile groups,or esterase labile groups, as disclosed in the above-identified patents,disulfide and thioether groups being preferred. Additional linkinggroups are described and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

2. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands”, U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

3. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ1I,α2I, α3I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al., Cancer Research53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998)and the aforementioned U.S. patents to American Cyanamid). Anotheranti-tumor drug that the antibody can be conjugated is QFA which is anantifolate. Both calicheamicin and QFA have intracellular sites ofaction and do not readily cross the plasma membrane. Therefore, cellularuptake of these agents through antibody mediated internalization greatlyenhances their cytotoxic effects.

4. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc99m or I123, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc⁹⁹m or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds expressly contemplate, but are not limited to, ADCprepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC,MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A). Seepages 467-498, 2003-2004 Applications Handbook and Catalog.

5. Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC), an antibody (Ab) is conjugated toone or more drug moieties (D), e.g. about 1 to about 20 drug moietiesper antibody, through a linker (L). The ADC of Formula I may be preparedby several routes, employing organic chemistry reactions, conditions,and reagents known to those skilled in the art, including: (1) reactionof a nucleophilic group of an antibody with a bivalent linker reagent,to form Ab-L, via a covalent bond, followed by reaction with a drugmoiety D; and (2) reaction of a nucleophilic group of a drug moiety witha bivalent linker reagent, to form D-L, via a covalent bond, followed byreaction with the nucleophilic group of an antibody. Additional methodsfor preparing ADC are described herein.

Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“SMCC’), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“STAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates may also be produced by modification of theantibody to introduce electrophilic moieties, which can react withnucleophilic substituents on the linker reagent or drug. The sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

D. Methods

1. Diagnostic Methods and Methods of Detection

In one aspect, antibodies of the invention are useful for detecting thepresence of Notch2 in a biological sample. The term “detecting” as usedherein encompasses quantitative or qualitative detection. In certainembodiments, a biological sample comprises a cell or tissue, such ascancerous tissue.

In one aspect, the invention provides a method of detecting the presenceof Notch2 in a biological sample. In certain embodiments, the methodcomprises contacting the biological sample with an anti-Notch2 antibody(e.g., an anti-Notch2 NRR antibody) under conditions permissive forbinding of the anti-Notch2 antibody to Notch2, and detecting whether acomplex is formed between the anti-Notch2 antibody and Notch2.

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of Notch2. In certain embodiments,the method comprises contacting a test cell with an anti-Notch2antibody; determining the level of expression (either quantitatively orqualitatively) of Notch2 by the test cell by detecting binding of theanti-Notch2 antibody to Notch2; and comparing the level of expression ofNotch2 by the test cell with the level of expression of Notch2 by acontrol cell (e.g., a normal cell of the same tissue origin as the testcell, or a cell that expresses Notch2 at levels comparable to such anormal cell, or an average level of expression among multiple controlcells), wherein a higher level of expression of Notch2 by the test cellas compared to the control cell indicates the presence of a disorderassociated with increased expression of Notch2. In certain embodiments,the test cell is obtained from an individual suspected of having adisorder associated with increased expression of Notch2. In certainembodiments, the disorder is a cell proliferative disorder, such as acancer or a tumor.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cancer, e.g., B-cell malignancies, melanoma, T-cellmalignancies (e.g., T-ALL), breast cancer, brain cancer, cervicalcancer, colon cancer, and pancreatic cancer.

Certain other methods can be used to detect binding of antibodies toNotch2. Such methods include, but are not limited to, antigen-bindingassays that are well known in the art, such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays,protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, antibodies are labeled. Labels include, but arenot limited to, labels or moieties that are detected directly (such asfluorescent, chromophoric, electron-dense, chemiluminescent, andradioactive labels), as well as moieties, such as enzymes or ligands,that are detected indirectly, e.g., through an enzymatic reaction ormolecular interaction. Exemplary labels include, but are not limited to,the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

In certain embodiments, antibodies are immobilized on an insolublematrix. Immobilization may entail separating an anti-Notch2 antibodyfrom any Notch2 that remains free in solution. This conventionally isaccomplished by either insolubilizing the anti-Notch2 antibody beforethe assay procedure, as by adsorption to a water-insoluble matrix orsurface (Bennich et al., U.S. Pat. No. 3,720,760), or by covalentcoupling (for example, using glutaraldehyde cross-linking), or byinsolubilizing the anti-Notch2 antibody after formation of a complexbetween the anti-Notch2 antibody and Notch2, e.g., byimmunoprecipitation.

It is understood that any of the above embodiments of diagnosis ordetection may be carried out using an immunoconjugate of the inventionin place of or in addition to an anti-Notch2 antibody.

2. Therapeutic Methods

An antibody of the invention may be used in, for example, in vitro, exvivo, and in vivo therapeutic methods. In one aspect, the inventionprovides methods for inhibiting Notch2 activity, i.e., Notch2 signaling,either in vivo or in vitro, the method comprising exposing a cell to ananti-Notch2 NRR antibody of the invention under conditions permissivefor binding of the antibody to Notch2. In certain embodiments, the cellis a cancer cell, e.g., a cancerous B-cell or a melanoma cell. In oneembodiment, an anti-Notch2 NRR antibody of the invention can be used forinhibiting Notch2 activity, the method comprising exposing Notch2 to ananti-Notch2 NRR antibody of the invention such that Notch2 activity isinhibited.

An anti-Notch2 NRR antibody of the invention may be used, e.g., for thetreatment of disorders associated with expression and/or activity ofNotch2, e.g., disorders associated with increased expression or activityof Notch2, or disorders in which expression or activity of Notch2contributes to a pathogenic state. A “disorder associated with increasedexpression or activity of Notch2” refers to a disorder in which Notch2expression or activity is significantly higher than normal.

In one aspect, an antibody of the invention is used to treat or preventcancer, e.g., B-cell malignancies, melanoma, T-cell malignancies (e.g.,T-ALL), breast cancer, brain cancer, cervical cancer, colon cancer, andpancreatic cancer. In certain embodiments, an anti-Notch2 NRR antibodyis used to treat a cancer, such as breast cancer, a B-cell malignancy ormelanoma.

In a further aspect, an antibody of the invention is used to treat orprevent a cancer, specifically a solid tumor containing cancer stemcells. Cancer stem cells are capable of proliferating to give rise toadditional cancer stem cells, as well as other tumor cell populations.See Al-Hajj et al., Proc. Nat'l Acad. Sci. USA 100: 3983-3988 (2003).Cancer stem cells are believed to contribute to the recurrence ofcancers and the resistance of cancers to drug therapies. Notch receptorshave been identified as cancer stem cell markers and as a target foreliminating cancer stem cells responsible for the formation andrecurrence of solid tumors. See WO 2008/091641. Such solid tumorsinclude but are not limited to breast, colon, pancreatic, prostate,lung, head and neck, rectal, and colorectal cancers.

In one aspect, the invention provides methods for treating a cancercomprising administering to an individual in need thereof an effectiveamount of an antibody of the invention. In certain embodiments, a methodfor treating a cancer comprises administering to an individual in needthereof an effective amount of a pharmaceutical formulation comprisingan antibody of the invention and, optionally, at least one additionaltherapeutic agent, such as those provided below.

Antibodies of the invention can be used either alone or in combinationwith other compositions in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent and/or adjuvant. In certain embodiments, ananti-Notch1 antibody (e.g., an anti-Notch1 NRR antibody) is administeredwith an additional therapeutic agent that inhibits or reduces alteredintestinal cell differentiation that would otherwise be induced by suchanti-Notch1 antibody. In certain embodiments, the additional therapeuticagent is dexamethasone or tamoxifen. Such combination therapy would beuseful, e.g., in treating angiogenic disorders, includingtumor-associated angiogenesis, and cancer. In certain embodiments, ananti-Notch2 antibody (e.g., an anti-Notch2 NRR antibody) is administeredwith an additional therapeutic agent, e.g., a chemotherapeutic agent.Such combination therapy would be useful, e.g., in treating cancer, suchas B-cell malignancies and melanoma.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with radiation therapy.

In one aspect, at least some of the antibodies of the invention can bindNotch2 from species other than human. Accordingly, antibodies of theinvention can be used to bind Notch2, e.g., in a mammalian cell cultureexpressing endogenous or recombinant Notch2, in humans, or in othermammals having a Notch2 with which an antibody of the inventioncross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesusmonkeys, pig, rat, or mouse).

In one embodiment, an antibody of the invention is used in a method forbinding Notch2 in an individual suffering from a disorder associatedwith increased Notch2 expression and/or activity, the method comprisingadministering to the individual the antibody such that Notch2 in theindividual is bound. In one embodiment, the Notch2 is human Notch2, andthe individual is a human individual. Alternatively, the individual canbe a mammal expressing Notch2 to which an antibody of the inventionbinds. Still further the individual can be a mammal into which Notch2has been introduced (e.g., by administration of Notch2 or by expressionof a transgene encoding Notch2).

An antibody of the invention can be administered to a human fortherapeutic purposes. Moreover, an antibody of the invention can beadministered to a non-human mammal expressing Notch2 with which theantibody cross-reacts (e.g., a primate, pig, rat, or mouse) forveterinary purposes or as an animal model of human disease. Regardingthe latter, such animal models may be useful for evaluating thetherapeutic efficacy of antibodies of the invention (e.g., testing ofdosages and time courses of administration).

An antibody of the invention (and any additional therapeutic agent oradjuvant) can be administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. Inaddition, the antibody is suitably administered by pulse infusion,particularly with declining doses of the antibody. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic.

The location of the binding target of an antibody of the invention maybe taken into consideration in preparation and administration of theantibody. When the binding target is an intracellular molecule, certainembodiments of the invention provide for the antibody or antigen-bindingfragment thereof to be introduced into the cell where the binding targetis located. In one embodiment, an antibody of the invention can beexpressed intracellularly as an intrabody. The term “intrabody,” as usedherein, refers to an antibody or antigen-binding portion thereof that isexpressed intracellularly and that is capable of selectively binding toa target molecule, as described, e.g., in Marasco, Gene Therapy 4: 11-15(1997); Kontermann, Methods 34: 163-170 (2004); U.S. Pat. Nos. 6,004,940and 6,329,173; U.S. Patent Application Publication No. 2003/0104402, andPCT Publication No. WO2003/077945. See also, for example, WO96/07321published Mar. 14, 1996, concerning the use of gene therapy to generateintracellular antibodies.

Intracellular expression of an intrabody may be effected by introducinga nucleic acid encoding the desired antibody or antigen-binding portionthereof (lacking the wild-type leader sequence and secretory signalsnormally associated with the gene encoding that antibody orantigen-binding fragment) into a target cell. One or more nucleic acidsencoding all or a portion of an antibody of the invention can bedelivered to a target cell, such that one or more intrabodies areexpressed which are capable of binding to an intracellular targetpolypeptide and modulating the activity of the target polypeptide. Anystandard method of introducing nucleic acids into a cell may be used,including, but not limited to, microinjection, ballistic injection,electroporation, calcium phosphate precipitation, liposomes, andtransfection with retroviral, adenoviral, adeno-associated viral andvaccinia vectors carrying the nucleic acid of interest.

In certain embodiments, nucleic acid (optionally contained in a vector)may be introduced into a patient's cells by in vivo and ex vivo methods.In one example of in vivo delivery, nucleic acid is injected directlyinto the patient, e.g., at the site where therapeutic intervention isrequired. In a further example of in vivo delivery, nucleic acid isintroduced into a cell using transfection with viral vectors (such asadenovirus, Herpes simplex I virus, or adeno-associated virus) andlipid-based systems (useful lipids for lipid-mediated transfer of thegene are DOTMA, DOPE and DC-Chol, for example). For review of certaingene marking and gene therapy protocols, see Anderson et al., Science256:808-813 (1992), and WO 93/25673 and the references cited therein. Inan example of ex vivo treatment, a patient's cells are removed, nucleicacid is introduced into those isolated cells, and the modified cells areadministered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). A commonlyused vector for ex vivo delivery of a nucleic acid is a retroviralvector.

In another embodiment, internalizing antibodies are provided. Antibodiescan possess certain characteristics that enhance delivery of antibodiesinto cells, or can be modified to possess such characteristics.Techniques for achieving this are known in the art. For example,cationization of an antibody is known to facilitate its uptake intocells (see, e.g., U.S. Pat. No. 6,703,019). Lipofections or liposomescan also be used to deliver the antibody into cells. Where antibodyfragments are used, the smallest inhibitory fragment that specificallybinds to the target protein may be advantageous. For example, based uponthe variable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993).

Entry of antibodies into target cells can be enhanced by other methodsknown in the art. For example, certain sequences, such as those derivedfrom HIV Tat or the Antennapedia homeodomain protein are able to directefficient uptake of heterologous proteins across cell membranes. See,e.g., Chen et al., Proc. Natl. Acad. Sci. USA (1999), 96:4325-4329.

When the binding target of an antibody is located in the brain, certainembodiments of the invention provide for the antibody to traverse theblood-brain barrier. Several art-known approaches exist for transportingmolecules across the blood-brain barrier, including, but not limited to,physical methods, lipid-based methods, stem cell-based methods, andreceptor and channel-based methods.

Physical methods of transporting an antibody across the blood-brainbarrier include, but are not limited to, circumventing the blood-brainbarrier entirely, or by creating openings in the blood-brain barrier.Circumvention methods include, but are not limited to, direct injectioninto the brain (see, e.g., Papanastassiou et al., Gene Therapy 9:398-406 (2002)), interstitial infusion/convection-enhanced delivery(see, e.g., Bobo et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080(1994)), and implanting a delivery device in the brain (see, e.g., Gillet al., Nature Med. 9: 589-595 (2003); and Gliadel Wafers™, GuildfordPharmaceutical). Methods of creating openings in the barrier include,but are not limited to, ultrasound (see, e.g., U.S. Patent PublicationNo. 2002/0038086), osmotic pressure (e.g., by administration ofhypertonic mannitol (Neuwelt, E. A., Implication of the Blood-BrainBarrier and its Manipulation, Vols 1 & 2, Plenum Press, N.Y. (1989)),permeabilization by, e.g., bradykinin or permeabilizer A-7 (see, e.g.,U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and 5,686,416), andtransfection of neurons that straddle the blood-brain barrier withvectors containing genes encoding the antibody (see, e.g., U.S. PatentPublication No. 2003/0083299).

Lipid-based methods of transporting an antibody across the blood-brainbarrier include, but are not limited to, encapsulating the antibody inliposomes that are coupled to antibody binding fragments that bind toreceptors on the vascular endothelium of the blood-brain barrier (see,e.g., U.S. Patent Application Publication No. 20020025313), and coatingthe antibody in low-density lipoprotein particles (see, e.g., U.S.Patent Application Publication No. 20040204354) or apolipoprotein E(see, e.g., U.S. Patent Application Publication No. 20040131692).

Stem-cell based methods of transporting an antibody across theblood-brain barrier entail genetically engineering neural progenitorcells (NPCs) to express the antibody of interest and then implanting thestem cells into the brain of the individual to be treated. See Behrstocket al. (2005) Gene Ther. 15 Dec. 2005 advanced online publication(reporting that NPCs genetically engineered to express the neurotrophicfactor GDNF reduced symptoms of Parkinson disease when implanted intothe brains of rodent and primate models).

Receptor and channel-based methods of transporting an antibody acrossthe blood-brain barrier include, but are not limited to, usingglucocorticoid blockers to increase permeability of the blood-brainbarrier (see, e.g., U.S. Patent Application Publication Nos.2002/0065259, 2003/0162695, and 2005/0124533); activating potassiumchannels (see, e.g., U.S. Patent Application Publication No.2005/0089473), inhibiting ABC drug transporters (see, e.g., U.S. PatentApplication Publication No. 2003/0073713); coating antibodies with atransferrin and modulating activity of the one or more transferrinreceptors (see, e.g., U.S. Patent Application Publication No.2003/0129186), and cationizing the antibodies (see, e.g., U.S. Pat. No.5,004,697).

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the antibody. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

It is understood that any of the above therapeutic methods may becarried out using an immunoconjugate of the invention in place of or inaddition to an anti-Notch2 antibody.

3. Assays

Anti-Notch2 NRR antibodies of the invention may be characterized fortheir physical/chemical properties and/or biological activities byvarious assays known in the art.

a) Activity Assays

In one aspect, assays are provided for identifying anti-Notch2antibodies (e.g., anti-Notch2 NRR antibodies) having biologicalactivity. Biological activity may include, e.g., inhibition or reductionof Notch2 activity, e.g., Notch2 signaling. Antibodies having suchbiological activity in vivo and/or in vitro are also provided.

In certain embodiments, an anti-Notch2 NRR antibody of the invention istested for its ability to inhibit generation of marginal zone B cells.An exemplary assay is provided in the Examples. In certain otherembodiments, an antibody of the invention is tested for its ability toinhibit expression of a reporter gene that is responsive to Notch2signaling. An exemplary assay is provided in the Examples.

b) Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc. In another aspect, competition assays may be used to identify anantibody (e.g., a monoclonal antibody) that competes with an antibody ofthe invention. In one embodiment, an antibody competes with Antibody D,Antibody D-1, Antibody D-2, or Antibody D-3 for binding to Notch2. Inone such embodiment, a competing antibody binds to the same epitope(e.g., a linear or a conformational epitope) that is bound by AntibodyD, Antibody D-1, Antibody D-2, or Antibody D-3. In another embodiment, acompeting antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by anti-NRR1 as described inExample B(5), i.e., an epitope comprising LNR-A, LNR-B and HD-C domainsof Notch1 NRR. Exemplary competition assays include, but are not limitedto, routine assays such as those provided in Harlow and Lane (1988)Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). Detailed exemplary methods for mapping anepitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.). Two antibodies are said to “bind to thesame epitope” if each blocks binding of the other by 50% or more.

In an exemplary competition assay, immobilized Notch2 NRR is incubatedin a solution comprising a first labeled antibody that binds to Notch2NRR (e.g., Antibody D, Antibody D-1, Antibody D-2, or Antibody D-3) anda second unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to Notch2 NRR. The secondantibody may be present in a hybridoma supernatant. As a control,immobilized Notch2 NRR is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody to Notch2NRR, excess unbound antibody is removed, and the amount of labelassociated with immobilized Notch2 NRR is measured. If the amount oflabel associated with immobilized Notch2 NRR is substantially reduced inthe test sample relative to the control sample, then that indicates thatthe second antibody is competing with the first antibody for binding toNotch2 NRR.

In one aspect, antibodies of the invention can be further characterizedby a series of assays including, but not limited to, N-terminalsequencing, amino acid analysis, non-denaturing size exclusion highpressure liquid chromatography (HPLC), mass spectrometry, ion exchangechromatography and papain digestion.

It is understood that any of the above assays may be carried out usingan immunoconjugate of the invention in place of or in addition to ananti-Notch2 NRR antibody.

E. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or combined with another composition effective fortreating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anantibody or immunoconjugate of the invention. The label or packageinsert indicates that the composition is used for treating the conditionof choice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody or immunoconjugate of the invention; and (b) asecond container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

IV. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

A. Materials and Methods

1. Generation of Anti-Notch2 NRR Antibodies

Library Sorting and Screening to Identify Anti-Notch2 NRR Antibodies

Human phage antibody libraries with synthetic diversities in theselected complementarity determining regions (H1, H2, H3, L3), mimickingthe natural diversity of human IgG repertoire, were used for panning.Fab fragments were displayed bivalently on the surface of M13bacteriophage particles. (See Lee et al., J. Mol. Biol. 340:1073-1093(2004).) Notch2 NRR fragments were expressed as secreted proteins fusedto epitope tags (FLAG or 6×His) using the baculovirus expression vectorsystem or 293T cells, purified to >90% purity using affinitychromatography and tested for lack of aggregation using lightscattering. The sequences of the Notch2 NRR antigens were as follows:

FLAG-Human-Notch2-NRR-6xHis: (SEQ ID NO: 73)KDDDDKGSGDVCPQMPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCRKGEQCVHTASGPRCFCPSPRDCESGCASSPCQHGGSCHPQRQPPYYSCQCAPPFSGSRCELYTAPPSTPPATCLSQYCADKARDGVCDEACNSHACQWDGGDCSLTMENPWANCSSPLPCWDYINNQCDELCNTVECLFDNFECQGNSKTCKYDKYCADHFKDNHCNQGCNSEECGWDGLDCAADQPENLAEGTLVIVVLMPPEQLLQDARSFLRALGTLLHTNLRIKRDSQGELMVYPYYGEKSAAMKKQRMTRRSLPGEQEQEVAGSKVFLEIDNRQCVQDSDHCFKNTDAAAALLASHAIQGTLSYPLVSVVSESLTPERTEFGLVPRGSGHHHHHH Mouse Notch2-NRR-FLAG:(SEQ ID NO: 74) ADVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCRRGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCELYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTLRCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECGWDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALMVYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDAAAALLASHAIQGTLSYPLVSVFSELESPRNARRAGSGDYKDDDDKENLYFQ

Nunc 96 well Maxisorp immunoplates were coated overnight at 4° C. withtarget antigen (10 μg/ml) and were blocked for 1 hour at roomtemperature with phage blocking buffer PBST (phosphate-buffered saline(PBS) and 1% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) tween-20).Antibody phage libraries VH (see, e.g., Lee et al., J. Immunol. Meth.284:119-132, 2004) and VH/VL (see Liang et al., J. Mol. Biol. 366:815-829, 2007) were added to antigen plates separately and incubatedovernight at room temperature. The following day antigen-coated plateswere washed ten times with PBT (PBS with 0.05% Tween-20), and boundphage were eluted with 50 mM HCl and 500 mM NaCl for 30 minutes andneutralized with an equal volume of 1 M Tris base (pH7.5). Recoveredphage were amplified in E. coli XL-1 Blue cells. During the subsequentselection rounds, incubation of antibody phage with the antigen-coatedplates was reduced to 2-3 hours, and the stringency of plate washing wasgradually increased.

After 4 rounds of panning, significant enrichment was observed. 96clones were picked each from VH and VH/VL library sorting to determinewhether they specifically bound to both human and murine Notch2 NRR. Thevariable regions of these clones were PCR sequenced to identify uniquesequence clones.

The affinities of phage antibodies were ranked using spot competitionELISA. The phage antibody IC50 values were further determined usingcompetitive phage-binding ELISA. Unique phage antibodies that bind bothhuman and murine Notch2 NRR were chosen and reformatted to full lengthIgGs for evaluation in in vitro cell assay.

Clones of interest were reformatted into IgGs by cloning VL and VHregions of individual clones into the LPG3 and LPG4 vector respectively,transiently expressing in mammalian CHO cells, and purifying with aprotein A column.

Construct Libraries for Affinity Improvement of Antibody D

Phagemid pW0703 (derived from phagemid pV0350-2b (Lee et al., J. Mol.Biol 340, 1073-1093 (2004)) displaying monovalent Fab on the surface ofM13 bacteriophage) served as the library template for grafting light(V_(L)) and heavy (V_(H)) chain variable domains of Antibody D from theVH/VL library for affinity maturation. Stop codons were thenincorporated in CDR-L3 of the library template. Soft randomizationstrategy was used for affinity maturation, which introduced a mutationrate of approximately 50% at the selected positions by using a poisonedoligonucleotide strategy with 70-10-10-10 mixtures of bases favoring thewild type nucleotides (Gallop et al., Journal of Medicinal Chemistry37:1233-1251 (1994)). Specifically residues at positions 28-32 ofCDR-L1, 50 and 53-55 of CDR-L2, 91-94 and 96 of CDR-L3, 28-35 of CDR-H1,50-58 of CDR-H2, 95-100 of CDR-H3, were targeted. Three differentlibraries with combinations of soft-randomized CDR loops, L1/L2/L3,L3/H1/H2 and L3/H3, were also constructed.

Phage Sorting Strategy to Generate Affinity Improvement

For affinity improvement selection, phage libraries were subjected toplate sorting for the first round, followed by five rounds of solutionsorting. The libraries were sorted against human and murine Notch2 NRRseparately. At the first round of plate sorting, three libraries weresorted against target coated plate (NUNC Maxisorp plate) separately withphage input about 3 O.D./ml in 1% BSA and 0.05% Tween 20 for 2 hours atroom temperature. For the following round of solution sorting, 1 O.D./mlphage propagated from the first round of plate sorting were incubatedwith 50 nM biotinylated target protein (the concentration is based onparental clone phage IC50 value) in 100 μl buffer containing 1%Superblock (Pierce Biotechnology) and 0.05% Tween20 for 30 minutes atroom temperature. The mixture was further diluted 10× with 1%Superblock, and 100 μl/well was applied to neutravidin-coated wells (5μg/ml) for 15 minutes at room temperature with gentle shaking to capturebiotinylated target bound phage. The wells were then washed withPBS-0.05% Tween 20 ten times. To determine background binding, controlwells containing phage with targets that were not biotinylated werecaptured on neutravidin-coated plates. Bound phage was eluted with 0.1NHCl for 20 minutes, neutralized by 1/10 volume of 1M Tris pH11, titered,and propagated for the next round. Next, four more rounds of solutionsorting were carried out together with two methods of increasingselection stringency. The first of which is for on-rate selection bydecreasing biotinylated target protein concentration from 10 nM to 0.5nM, and the second of which is for off-rate selection by adding excessamounts of non-biotinylated target protein (100˜500 fold more) tocompete off weaker binders either at room temperature. Also, the phageinput was decreased (0.1˜0.5 O.D/ml) to lower background phage binding.

High Throughput Affinity Screening ELISA (Single Spot Competition)

Colonies were picked from the sixth round sorting and grown overnight at37° C. in 500 μl/well of 2YT media with 50 μg/ml carbenicillin and1E10/ml KO7 in 96-well plate (Falcon). From the same plate, a colony ofXL-1 infected parental phage was picked as control. 96-well NuncMaxisorp plates were coated with 100 μl/well of human and murine Notch2NRR protein (1 μg/ml) separately in PBS at 4° C. overnight or roomtemperature for 2 hours. The plates were blocked with 65 μl of 1% BSAfor 30 min and 40 μl of 1% Tween 20 for another 30 minutes.

The phage supernatant was diluted 1:5 in ELISA (enzyme linkedimmunosorbent assay) buffer (PBS with 0.5% BSA, 0.05% Tween20) with orwithout 10 nM target protein in 100 μl total volume and incubated atleast 1 hour at room temperature in an F plate (NUNC). 75 μl of mixturewith or without target protein was transferred side by side to thetarget protein coated plates. The plate was gently shaken for 15 min toallow the capture of unbound phage to the target protein-coated plate.The plate was washed at least five times with PBS-0.05% Tween 20. Thebinding was quantified by adding horseradish peroxidase (HRP)-conjugatedanti-M13 antibody in ELISA buffer (1:5000) and incubated for 30 minutesat room temperature. The plates were washed with PBS-0.05% Tween 20 atleast five times. Next, 100 μl/well of a 1:1 ratio of3,3′,5,5′-tetramethylbenzidine (TMB) Peroxidase substrate and PeroxidaseSolution B (H₂O₂) (Kirkegaard-Perry Laboratories (Gaithersburg, Md.))was added to the well and incubated for 5 minutes at room temperature.The reaction was stopped by adding 100 μl 1M Phosphoric Acid (H₃PO₄) toeach well and allowed to incubate for 5 minutes at room temperature. TheOD (optical density) of the yellow color in each well was determinedusing a standard ELISA plate reader at 450 nm. The OD reduction (%) wascalculated by the following equation.

OD_(450 nm) reduction (%)=[(OD_(450 nm) of wells withcompetitor)/(OD_(450 nm) of well with no competitor)]*100

In comparison to the OD_(450 nm) reduction (%) of the well of parentalphage (100%), clones that had the OD_(450 nm) reduction (%) lower than50% for both the human and murine target were picked for sequenceanalysis. Unique clones were selected for phage preparation to determinebinding affinity (phage IC50) against both human and murine Notch2 NRRby comparison with parental clones. The clones with the most affinityimprovement were reformatted into human IgG1 and expressed in Mammaliancells.

Characterization of Anti-Notch2 NRR Antibodies (Biacore)

Binding affinities of anti-Notch2 NRR antibodies were measured bySurface Plasmon Resonance (SRP) using a BIAcore™-3000 instrument.Anti-Notch2 NRR human IgGs were captured by mouse anti-human IgG coatedon the CM5 sensor chip to achieve approximately 200 response units (RU).For kinetics measurements, two-fold serial dilutions of human and mouseNotch2 NRR (3.9 nM to 500 nM) were injected in PBT buffer (PBS with0.05% Tween 20) at 25° C. with a flow rate of 30 μl/min. Associationrates (k_(on)) and dissociation rates (k_(off)) were calculated using asimple one-to-one Langmuir binding model (BIAcore Evaluation Softwareversion 3.2). The equilibrium dissociation constant (K_(D)) wascalculated as the ratio k_(off)/k_(on).

2. Generation of Anti-Notch1 NRR Antibodies

The generation and characterization of certain anti-Notch1 NRRantibodies have been previously described. See U.S. Patent ApplicationPublication No. US 2009/0081238 A1.

3. ELISA

Anti-Flag antibody was used to capture Flag-Tagged NRR protein from allfour Notch receptors in a black walled, high binding plate (Greiner).NRR antibody and E25 control antibodies were bound to the NRR proteinsand washed away with PBS. An Alkaline Phosphate conjugated anti-human(H+L) secondary antibody (Jackson ImmunoResearch) was used to detectbound anti-NRR and E25. Unbound secondary was washed away and AP wasdetected with PNPP substrate (Pierce).

4. FACS

Expression plasmids were made in which either the Notch1 or Notch2extracellular domain and trans-membrane domains were N-terminallymyc-tagged and expressed under the control of a Tetracycline ResponseElement (TRE) upstream of a minimal CMV immediate early promoter. Theseplasmids were stably transfected into CHO-K1 cells (Clonetech),engineered to express a transactivator protein in the presence ofDoxycycline that binds to and activates the TRE in the transfectedplasmid. Cells were treated with 1 mg/mL doxycycline or vehicle for 48hrs, and then stained with anti-NRR, anti-myc (9E10, Millipore) or E25control antibodies and either goat anti-human Alexa 488 or Alexa 647secondary antibodies (Invitrogen). After staining, cells were run on aFACScalibur machine (BD Biosciences) and 15000 events were collected andrange gates were drawn to assess positively and negatively stainedpopulations.

5. Notch Reporter Assay

NIH-3T3 cells either stably transfected with Notch1 or transientlytransfected with plasmids containing other Notch receptors wereco-transfected with a Notch-responsive TP-1 (12×CSL) Firefly luciferasereporter and a constitutively active Renilla Luciferase reporter(pRL-CMV, Promega) to control for transfection efficiency. Cells areallowed to recover from the transfection from 6 hours to overnight.Treatments of antibodies and NIH-3T3 cells stably transfected withligand are used to stimulate the receptor cells. After 20 hours, fireflyand Renilla luciferase are measured with Dual Glo Luciferase Assaysystem (Promega). Eight replicates were analyzed for each condition bydividing the Firefly signal by the Renilla signal to control fortransfection efficiency. The mean and standard deviation were calculatedand values were normalized to calculated values for co-culturestimulated with NIH-3T3 cells without ligand transfected.

6. Epitope Mapping

Synthetic Notch1 and Notch2 NRR sequences were designed with uniquerestriction sites at junctions between the five domains of theNRR—LNR-A; LNR-B; LNR-C; HD-N; and HD-C—and produced by Blue HeronTechnology (Bothell, Wash.). Swapping various domains between the twosynthetic sequences, using the engineered restriction sites, generatedchimeric NRR proteins. The chimeric clones were assembled in a modifiedpUC19 vector and transferred to a SEAP containing vector, pCSC.AP, forexpression. AP-tagged NRR proteins were produced by transfection of 293Tcells. After 72 hr culture, the conditioned media was collected, clearedby centrifugation, and tested for AP activity using the Phospha-LightSystem (Applied Biosystems, Bedford, Mass.). The AP activity in eachsample was normalized and an ELISA was performed to test the ability ofα-NRR1 or α-NRR2 to bind the chimeric NRR proteins. The ELISA wasperformed as follows: either α-NRR1 or α-NRR2 was captured by a platebound a-Fc antibody in 96-well format and incubated with the conditionedmedia overnight. The plate was washed to remove unbound protein, and thecaptured protein was tested for AP activity using the Phospha-LightSystem. The resulting AP activity was used to quantify the capacity ofeach α-NRR antibody to bind specific chimeric proteins and inferimportant epitopes for binding.

7. HUVEC Fibrin Gel Bead Assay

HUVEC cells were grown on fibrin gel beads as previously described(Nakatsu et al., 2003). Cytodex 3 beads (Amersham Pharmacia Biotech)were coated with 350-400 HUVECs per bead in 2 ml EGM-2 medium(Clonetics). About 200 HUVEC coated beads were imbedded in fibrin clotin one well of 12-well tissue culture plate. 6×10⁴ Detroit 551fibroblast cells were plated on top of the clot and medium containingeither 5 ug/ml anti-NRR1, 5 ug/ml anti-DLL4, 1 uM DBZ, or vehiclecontrols. Medium was changed every two days. After seven to nine days,fibrin gels were then fixed with 4% paraformaldehyde and endothelialcells were stained with anti-CD31 (R&D Systems AF806) using aFITC-conjugated secondary antibody for detection.

8. Neonatal Retinal In Vivo Assay

CD1 neonates were injected i.p. at P1 and P3 with either 10 mg/kg ofragweed control or 20 mg/kg of Notch-1. Eyes were harvested at P5 andfixed with 4% PFA. The retinas were dissected out, blocked (5% BSA, 0.5%Triton X-100) for one hour followed by treatment for 10 min with 1MSodium Citrate. Retinas were then incubation overnight at 4 C withbiotinylated isolectin B4 (Sigma) and Ki67 antibody (NeoMarkers).Retinas were subsequently washed and stained with streptavidin Alexa 488and goat anti-rabbit Cy3 in 1% BSA, 0.5% Triton X-100. Retinas weremounted flat and imaged using an epifluoresence microscope.

9. In Vivo Tumor Studies

The human tumor cell line Calu-6 and HM7 were grown in Ham's F12, lowglucose DMEM 1:1 supplemented with 10% v/v FBS, 1% v/vpenicillin/streptomycin, 2 mM L-Gln and 1 μg/ml Fungizone™ (Invitrogen™,CA). Cells were incubated at 37° C. in an atmosphere of 95% air/5% CO₂.For mouse xenograft experiments, tumor cells were suspended at aconcentration of 1×10⁸ or 1×10⁹ cells/ml and injected (100 μl/mouse)subcutaneously into the dorsal flank of Balb-c nude mice (Harlan SpragueDawley, IN). When tumors reached a volume of 400 mm³, a cohort wasrandomly selected (n=10) as day-0 controls. The remaining mice weredivided into groups of 10 mice, and phage antibody Anti-Notch1 wereadministered i.p. at the dose of 10 mg/kg twice weekly. The transplantedtumors were measured twice weekly along the longest axis and theperpendicular axis as described. For each day on which tumors weremeasured, the tumor volume for each mouse was calculated, and the tumorvolume means from each control antibody (anti-Ragweed), anti-VEGF groupwere compared with the tumor volume mean of Anti-Notch1-treated mice byDunnett's t test implemented in the JMP™ Statistical Analysis System(version 5.1 for Windows; SAS Institute, Cary, N.C.), at a level ofP<0.05. Mice were killed when tumor volume reached 2,000 mm³. Tumorswere sectioned by cryostat to 7 microns, fixed with acetone and stainedwith DAPI (Invitrogen), hamster anti-CD31 (Serotech, Inc.) and ananti-hamster Cy3 secondary (Jackson Immunolabs). Slides were mountedwith Fluorescent Mounting Medium (Dako). Images were taken with a ZeissAxioskop2 Microscope and analyzed by ImageJ for area of DAPI stain andCy3 staining. Cy3 stain was divided by the area of DAPI stain tonormalize to the number of cells present and the values were normalizedto the anti-Ragweed treated tumor. All animal studies were conducted inaccordance with Institutional Animal Care and Use Committee approvedprotocols.

10. Marginal Zone B Cell Analysis

Balb-c mice (Jackson) at 12 weeks old were injected IP twice per weekfor two weeks with 5 mg/kg of anti-gD, anti-NRR1 or anti-NRR2.Splenocytes were isolated from the mice and were stained for FACS withB220-PerCP, CD23-PE and CD21-FITC (BD Biosciences) as previouslydescribed (Saito, et al., Immunity, Vol. 18, 675-685, May 2003).

11. Histology and Immunohistochemistry of Mouse Intestine Tissue

Six groups of six, 10 week old, C57BL/6 mice (Jackson) were administeredeither MCT every other day, DBZ at 30 umol/kg every other day, Anti-NRR1at 10, 2, or 0.4 mg/kg or Anti-gD control (HSV-1) at 10 mg/kg at Day 0,2 and 6, or as otherwise indicated. Formalin-fixed and paraffin-embeddedmouse small intestine tissues were sectioned at 3 micron thickness.Histochemical identification of intestinal cell types was performed withAlcian blue as recommended by the manufacturer (PolyScientific). Foranti-Ki67 staining, sections were pretreated with Target RetrievalSolution (S1700, DAKO), and incubated with rabbit anti-Ki67 (1:200,clone SP6, Neomarkers). Secondary goat anti-rabbit at 7.5 μg/ml (Vectorlabs) was detected with the Vectastain ABC Elite Kit (Vector labs). Ki67stained sections were counterstained with Mayer's haemotoxylin. Foranti-lysozyme staining, sections were processed on the Discovery XTplatform (Ventana Medical Systems, Inc.) using CC1m epitope recoveryconditions, OmniMap-rabbit detection and Ventana haemotoxylin II/withbluing counterstain. For HES-1 staining, anti-rat HES-1 (clone NM1, MBL,International) was followed by TSA-HRP.

B. Results

1. Generation of Synthetic Antibodies that Specifically Target theNotch1 and Notch2 Negative Regulatory Regions

To enable independent antagonism of Notch1 and Notch2 signaling inhumans and mice, we used phage display to target the NRR and select forsynthetic, human antibodies with the following qualities. First, eachantibody was selected to potently inhibit its cognate target - - -Notch1 or Notch2 - - - but not other Notch receptors. Second, theantibodies were selected for binding to human target sequences to enabletherapeutic targeting. Third, in order to discover the biologicalconsequences of systemically inhibiting Notch1 or Notch2 in vivo and tostudy the functions of these receptors in mouse models, the antibodieswere co-selected for binding to the orthologous mouse sequences. Fourth,to avoid a human anti-antibody immune response, the complementaritydetermining regions were cloned into a human immunoglobulin framework.

Following this approach, we successfully isolated anti-Notch1 NRR andanti-Notch2 NRR antibodies with each of these desired properties. Theseantibodies have several advantages over “pan-Notch” inhibitors thattarget multiple Notch receptors, such as γ-secretase inhibitors. Becausethese antibodies distinguish individual Notch receptors, they candissect the distinct functions of those individual receptors.Furthermore, pan-Notch inhibitors show potentially unwanted side effectsin vivo due to the dual inhibition of both Notch1 and Notch2, and thusantibodies that are selective for either Notch1 or Notch2 may be moreattractive candidates for therapeutic purposes. The anti-Notch1 NRR andanti-Notch2 NRR antibodies isolated according to the above approach aredescribed and characterized in further detail below:

The isolation and characterization of anti-Notch NRR1 antibodies arediscussed in U.S. Patent Application Publication No. US 2009/0081238 A1.One of the antibodies disclosed in that application, “Antibody A-2,” wascharacterized with respect to its structure and certain biologicalactivities. Certain studies discussed in that application arerecapitulated herein. For convenience, “Antibody A-2” from U.S. PatentApplication Publication No. US 2009/0081238 A1 is referred to herein as“anti-NRR1.”

The isolation and characterization of anti-Notch2 NRR antibodies isdescribed herein. An anti-Notch2 NRR antibody, called “Antibody D”, wasisolated. That antibody was affinity matured as described above,resulting in Antibody D-1, Antibody D-2, and Antibody D-3. FIG. 1 showsthe HVR-H1, HVR-H2, and HVR-H3 sequences of Antibody D, Antibody D-1,Antibody D-2, and Antibody D-3. FIG. 2 shows the HVR-L1, HVR-L2, andHVR-L3 sequences of Antibody D, Antibody D-1, Antibody D-2, and AntibodyD-3. FIG. 3 shows the heavy chain variable region sequences of AntibodyD, Antibody D-1, Antibody D-2, and Antibody D-3. FIG. 4 shows the lightchain variable region sequences of Antibody D, Antibody D-1, AntibodyD-2, and Antibody D-3. For convenience, Antibody D-3 is referred toherein as “anti-NRR2.”

Measurements of binding affinities using surface plasmon resonance (SPR)revealed that anti-NRR1 bound with similar and high affinities (K_(d)=3nM) to purified NRR1 proteins of either mouse or human sequence. Weobserved a similar affinity (K_(d)=5 nM) to the NRR2 antigens in testsof anti-NRR2. Binding of both antibodies appeared highly specific forthe cognate receptor; no binding to the human or mouse sequences of anyof the other three non-targeted receptors was detected for eitheranti-NRR1 or anti-NRR2 using a variety of techniques, including SPR,enzyme-linked immunosorbent assay (ELISA) and fluorescence-activatedcell sorting (FACS) (FIG. 9 and data not shown). For example, ELISAresults showed that anti-NRR1 bound equally well to purified human andmouse Notch1-NRR proteins, with half-maximal binding observed at anantibody concentration of approximately 0.1 μg/ml. Anti-NRR1 did notdetectably bind to purified human or mouse Notch2 NRR, Notch3-NRR orNotch4-NRR proteins, even at the highest antibody concentration tested(FIG. 9A, left panel), despite the fact that human Notch1-NRR and humanNotch2 NRR sequences are 45% identical (FIG. 18). We observed similarresults for anti-NRR2, which bound specifically to the Notch2 NRRproteins but not to the NRR proteins from the other Notch receptors(FIG. 9A, right panel).

To determine whether anti-NRR1 and anti-NRR2 also bind specifically tofull-length receptors expressed on the surface of cells, we usedfluorescence-activated cell sorting (FACS). We assessed binding toeither Notch1 or Notch2, each carrying a myc epitope tag at theN-terminus to distinguish the transgenic Notch receptors fromendogenously expressed Notch receptors (particularly Notch2, which wasexpressed by the K1-CHO cells). Receptor expression was induced inK1-CHO cells that were either left untransfected (FIG. 9B, Panels 1, 4,7 and 10), transfected with myc-Notch1 (FIG. 9B, Panels 2, 5, 8 and 11)or transfected with myc-Notch2 (FIG. 9B, Panels 3, 6, 9 and 12). Bindingof anti-NRR1 to myc-positive cells was observed in the myc-N1 cells(FIG. 9B, panel 2), but not in the K1-CHO parent cells (FIG. 9B,panel 1) and only following induction (FIG. 9B, compare panels 2 and 5),indicating that anti-NRR1 bound specifically to Notch1. In contrast,although myc-Notch2 expression was clearly induced in the myc-N2 cells(FIG. 9B, compare panels 3 and 6), anti-NRR1 did not bind to myc-Notch2(FIG. 9B, compare panels 2 and 3). We performed a similar analysis usinganti-NRR2 (FIG. 9B, panels 7-12), although this analysis was complicatedby leaky expression of myc-Notch2 in the absence of induction (FIG. 9B,panels 6 and 12) and endogenous expression of hamster Notch2 in theK1-CHO line (FIG. 9B, compare panels 7 and 10 to panels 1 and 4).Nevertheless, anti-NRR2 significantly bound only to cells expressingmyc-Notch2 after induction, consistent with specific binding to Notch2(FIG. 9B, compare panel 9 to panels 8 and 12). Taken together, ourbinding results using purified NRR proteins as well as cell-surfaceexpression of full-length receptors demonstrate that anti-NRR1 bindsspecifically to Notch1-NRR1 and not to Notch2 NRR, and likewise, thatanti-NRR2 binds specifically to Notch2 NRR and not to Notch1-NRR. Thisspecificity is further supported by the numerous structural andfunctional studies described below.

2. Anti-NRR1 and Anti-NRR2 Function as Potent and Specific Inhibitors ofNotch1 and Notch2 Signaling In Vitro

To assess whether the anti-NRR antibodies affected Notch signaling, wefirst used a co-culture assay that employed NIH-3T3 cell linesengineered to express Jag1, as the Notch ligand, or either Notch1 (FIG.10A) or Notch2 (FIG. 10B) as the Notch receptor. The assays accuratelyreflected Notch signaling because a strong Notch reporter signal(Firefly luciferase) depended on the presence of both ligand- andreceptor-expressing cells (FIGS. 10A and B, compare −Jag1 to +Jag1), andthe reporter level was reduced to background when a GSI (γ-secretaseinhibitor) was included (FIGS. 10A and B, compare DMSO to DAPT).

Addition of increasing amounts of anti-NRR1 inhibited signaling in theNotch1 cells, with complete inhibition observed at an antibodyconcentration between 80 and 400 ng/ml (FIG. 10A). A control humanantibody failed to inhibit signaling (FIG. 10A, compare α-gD to α-NRR1titration), as did numerous other antibodies directed to antigens otherthan Notch1 (data not shown). To further test whether this inhibitoryactivity reflected anti-NRR1 binding, we asked whether the addition ofpurified NRR1 antigen rescued signaling in the presence of an inhibitoryconcentration (80 ng/ml) of anti-NRR1 antibody, by competing forantibody binding to Notch1 expressed on the signaling cells. Addition ofNRR1 but not NRR2 antigen (which appeared active and properly folded inother assays; see FIG. 10B) restored signaling to control levels, (FIG.10A, compare 80 ng/ml α-NRR1+NRR1 to 80 ng/ml α-NRR1 alone and 80 ng/mlα-NRR1+NRR2). As a control, neither NRR1 nor NRR2 antigens affectedsignaling in co-cultures that contained a control antibody, confirmingthat the NRR fragments themselves have no direct effect on Notchsignaling (FIG. 10A, α-gD+NRR1 and α-gD+NRR2). Using the Notch2expressing cells to assay anti-NRR2 activity, we observed similarresults for anti-NRR2 inhibition of Notch2 signaling (FIG. 10B). Bothantibodies inhibited signaling induced through all of the Notch ligandsthat we have tested, namely Jag1, Jag2, Dll1 and Dll4 (FIG. 16 and datanot shown). Taken together, these results demonstrate that anti-NRR1 andanti-NRR2 are potent paralog-specific inhibitors of signaling fromNotch1 and Notch2, respectively.

3. Anti-NRR1 Inhibits Signaling Through Both of the Main Classes ofMutant Receptors Found in T-ALLs

One mechanism through which Notch signaling has been shown to directlyaffect oncogenesis is through mutational activation of Notch1 signalingin T-ALL. In particular, Notch1 activating mutations in T-ALL fall intotwo broad categories: (1) those that truncate the PEST domain, thusstabilizing the ICD and increasing signaling in a ligand-dependentmanner, and (2) those that destabilize the NRR, thus enabling ADAMcleavage and stimulating signaling in a ligand-independent manner. Totest whether anti-NRR1 can abrogate signaling through such mutantreceptors, we modified the co-culture assay to express Notch1-WT (Notch1wild-type), Notch1-ΔPEST (Notch1 lacking the PEST domain), orNotch1-L1594P (Notch1 carrying the L1594P mutation in theheterodimerization (HD) domain of the NRR). As was observed forwild-type Notch1 signaling (FIG. 10C, top panel), anti-NRR1 completelyinhibited ligand-dependent and -independent signaling throughNotch1-ΔPEST and Notch1-L1594P (FIG. 10C, middle and bottom panels).Thus, anti-NRR1 antagonizes signaling through both classes of mutantreceptors, including the L1594P mutation that falls within anddestabilizes the same domain targeted by the antibody.

4. Anti-NRR1 and Anti-NRR2 Function as Potent and Specific Inhibitors ofNotch1 and Notch2 Signaling In Vivo

To determine whether anti-NRR1 and anti-NRR2 function asreceptor-specific inhibitors in vivo, we investigated how the antibodiesaffected cell fate decisions for which genetic techniques hadestablished a role for Notch1 or Notch2. Specifically, conditionalinactivation of Notch1 or Notch2 previously revealed that Notch1 is keyto determining a T versus B cell fate during lymphoid development,whereas Notch2 is required to generate splenic marginal zone B (MZB)cells. Consistent with a crucial function for Notch1 in T celldevelopment, we found that treating mice with anti-NRR1, but notanti-NRR2, significantly reduced thymus weights (FIGS. 11A and B).Likewise, anti-NRR1, but not anti-NRR2, dramatically reduced the totalnumber of cells in the thymus (FIG. 11B) and nearly completely inhibitedthe generation of CD4+/CD8+ double-positive T cells (FIG. 11C).

In contrast, the antibody effects were reversed when MZB cells wereexamined. Treatment with anti-NRR2 nearly eliminated MZB cells(0.97+/−0.45 compared to the control value of 6.61+/−0.25), reducing thepopulation even more dramatically than did purified, recombinantlymphotoxin-β receptor (LTβR)-Fc fusion protein (3.48+/−0.06), whichserved as a positive control (FIG. 11D). This inhibitory effect wasspecific to anti-NRR2 because anti-NRR1 did not significantly reduce theMZB population (6.00+/−0.44) relative to the anti-gD control antibody(6.61+/−0.25; FIG. 11D). Taken together, these in vivo studies indicatethat both antibodies are potent inhibitors of their respective targetsthat can induce biologically significant changes in cell fate decisions;furthermore, because anti-NRR1 affects the generation of T but not MZBcells whereas anti-NRR2 affects the generation of MZB but not T cells,each antibody functions specifically for its intended target in vivo.

5. A Co-Crystal Structure Reveals that Anti-NRR1 Bridges the LNR andHD-C Domains and Likely Stabilizes the NRR “Off” Conformation

To elucidate the binding specificity of anti-NRR1 and anti-NRR2 and gainmechanistic insights into the antibody antagonist activity, we testedbinding of both antibodies to a battery of chimeric NRR proteins.Specifically, we made chimeric NRRs by swapping each of the NRRsubdomains (LNR-A, LNR-B, LNR-C, HD-N and HD-C) between Notch1 andNotch2, expressed these chimeras as secreted proteins fused to alkalinephosphatase for easy detection, and assayed binding of normalizedamounts of each chimeric NRR to anti-NRR1 or anti-NRR2. Of the 26chimeric NRRs that we expressed, 17 were efficiently detected assecreted proteins (FIG. 12A), 7 were weakly detected and 2 were notdetected above background (FIG. 17), suggesting that most but not all ofthe chimeric NRRs were expressed, properly folded and secreted. Swappingthe single LNR-A domain from NRR2 into the NRR1 backbone disruptedbinding of anti-NRR1 (FIG. 12A, chimera BC.Hd and all others with LNR-Afrom NRR2), indicating that anti-NRR1 contacts with the LNR-A subdomainof NRR1 are essential for binding. In contrast, swapping the LNR-Cand/or HD-N domains from NRR2 into NRR1 did not affect anti-NRR1 binding(FIG. 12A, chimeras AB.Hd, ABC.Hc and AB.Hc), demonstrating that neitherof these domains is essential for binding specificity. Lastly, AB.Hc wasthe chimera with the fewest NRR1 subdomains that still supported fullanti-NRR1 binding (although weak binding was observed to the ABchimera), suggesting that most, and perhaps all, of the contactsrequired for anti-NRR1 binding specificity are contained in the LNR-A,LNR-B and HD-C domains.

Characterization of anti-NRR2 revealed a binding pattern similar to thatdefined for anti-NRR1, albeit with some distinct differences. Swappingthe LNR-A domain of NRR1 into the NRR2 backbone completely disruptedanti-NRR2 binding, whereas swaps of LNR-B or LNR-C had no effect; thus,as was the case for anti-NRR1, anti-NRR2 binding requires the LNR-Asubdomain from Notch2. Likewise, the HD-C domain from Notch2 was alsoessential. Two different chimeras (C.Hn and BC) with only threesubdomains from NRR2 supported full binding in this assay; both of thesechimeras share the LNR-A and HD-C subdomains from NRR2 and, consistentwith this observation, the chimera that contained only the LNR-A andHD-C subdomains from NRR2 supported weak but detectable binding toanti-NRR2. Thus, in defining the subdomains that determine anti-NRR2binding specificity, the domain swap experiments: (a) reveal that theLNR-A and HD-C are most important (necessary and partially sufficientfor binding specificity) and (b) suggest that antibody-NRR2 contacts inLNR-B and HD-N may also play a role.

To better understand the molecular basis of the antagonistic activity ofanti-NRR1, we determined the 2.2 Å crystal structure of the Fab fragmentof the antibody bound to human NRR1. This structure revealed that NRR1forms a compact structure very similar to that of human NRR2, with threeCa²⁺ binding LNR modules wrapped around the core HD domain (FIGS. 12Band C). The structure reveals that the apparent affect of Fab binding isto stabilize the LNR-HD interactions such that S2 is not accessible forcleavage, thus keeping the Notch1 signaling cascade quiescent. Thisinterpretation is supported by the observation that the Fab does notdirectly occlude the S2 site but instead binds at the interface betweenthe HD, LNR-A and LNR-B domains (FIG. 12D), consistent with the domainswap experiments (FIG. 12A). This interface buries ˜2000 Å² ofsolvent-accessible surface area, equally divided between the fab andNRR1. The Fab heavy chain contacts LNR-B, the final helix in the HD-Cdomain, and the periphery of LNR-A. CDR H3 nestles into the interfacebetween LNR-B and HD. In particular, R99 from H3 forms a hydrogen bondto the backbone carbonyl of Phe1501 from LNR-A while the aliphaticportion of the side chain interacts with L1710 from the HD domain. Thelight chain accounts for ˜40% of the buried surface area contributed bythe Fab and contacts the connecting loop prior to the final helix in theHD domain as well as LNR-A.

This structure also reveals the basis of Fab specificity for Notch1 overNotch2. Despite the ˜45% sequence identity between the NRR domains ofNotch1 and Notch2, most of the residues involved in the interface aredistinct between the two proteins. For instance, of the 21 NRR residuesthat bury at least 25% of solvent accessible surface area on bindingFab, only six are identical in Notch1 and Notch2. These six residuescontribute less than a quarter of the surface area in the Fab epitopeand are distributed throughout the epitope (FIGS. 12D and 18).

6. Selective Inhibition of Notch1 Signaling Deregulates Angiogenesis

To begin to exploit Notch receptor-specific antibodies to define thefunctional importance of individual receptors in vivo, we first askedwhether selectively blocking Notch1 signaling with anti-NRR1 woulddisrupt mammalian angiogenesis. Numerous reports indicate that the Notchpathway functions downstream of vascular endothelial growth factor(VEGF) and plays a key role in angiogenesis by regulating endothelialcell fate choice between tip and stalk cells. Experiments using avariety of genetic and biochemical tools, including Dll4-specificblocking antibodies, have established Dll4 as the key Notch ligandinvolved in Notch angiogenic signaling, although two Notch receptors,Notch1 and Notch4, have been implicated as receptors for Dll4 inangiogenesis.

We first tested whether selective inhibition of Notch1 affectedendothelial sprouting in vitro. Human umbilical vein endothelial cells(HUVECs) co-cultured with human skin fibroblasts generate sprouts withlumen-like structures. Consistent with previous work, we found thatpan-Notch inhibition using DBZ, an inhibitor of γ-secretase, as well asselective antibody blocking of Dll4 increased both the number and lengthof endothelial cell sprouts. Importantly, selective blocking of Notch1signaling using anti-NRR1 generated a similar phenotype (FIGS. 13A and13B).

To determine whether anti-NRR1 affects angiogenesis in vivo, we used amodel based on development of retinal vasculature in mouse neonates.Systemic delivery of anti-NRR1 dramatically disrupted development of theretinal vasculature in treated neonate mice (FIG. 13C). Relative totreatment with a control anti-ragweed antibody, anti-NRR1 treatmentgenerated a dense, compact and seemingly tangled vascular network (FIG.13C, compare panels I and III with panels II and IV, respectively). Thisincreased accumulation of endothelial cells correlated with increasedproliferation, as assessed by labeling with the Ki67 proliferationmarker (FIG. 13C, compare panels V and VI). These observations,including increased vascular density, endothelial cell accumulation andincreased proliferation, all closely mimic those found followinginhibition of Dll4 or γ-secretase using this same neonate model (datanot shown) and are consistent with results from other studies, includinganalysis of tumor vasculature (FIGS. 13D and 13E) and the mouse cornealpocket assay (data not shown). Our results demonstrate not only thatanti-NRR1 possesses potent biological activity but also that inhibitionof Notch1 alone, as opposed to Notch4 or a combination of Notch1 andNotch4, is sufficient to dramatically disrupt mammalian angiogenesis.

7. Selective Antibody Blocking of Notch1 Inhibits Tumor Growth inPreclinical Models

To determine whether selectively blocking Notch1 using anti-NRR1 wouldbe sufficient to inhibit tumor growth, we used preclinical tumor modelsand compared growth of established tumors following treatment withanti-NRR1, anti-VEGF or anti-ragweed antibodies. In both the Calu6 andHM7 models, the groups treated with anti-NRR1 showed a significantdecrease in tumor size relative to the control groups, even at the firsttime points examined, three to four days after the initial dose (FIGS.13A-C). In the Calu6 model, the anti-NRR1 group showed a decrease intumor size, from approximately 250 mm³ to less than 100 mm³, similar tothe decrease observed in the anti-VEGF group; in contrast, tumors in theanti-ragweed control group grew to a size greater than 400 mm³ (FIG.14A). We observed similar results using the aggressively growing HM7model; anti-NRR1 treatment caused tumor size to remain static (FIG. 14B)or nearly static (FIG. 14C) over 12-13 days, a dramatic differencerelative to the six-day growth from 250 mm³ to greater than 900 mm³ thatwas observed in the control group (FIGS. 14B and 5C). We observedsimilar inhibition of tumor growth using a range of anti-NRR1 doseconcentrations, between 20 mg/kg and 2.5 mg/kg, although the 10 and 20mg/kg groups showed a trend towards stronger inhibition relative to the2.5 and 5 mg/kg groups (FIG. 14C).

We investigated the Calu6 or HM7 tumor models because their response toanti-angiogenic agents has been well documented. To directly testwhether anti-NRR1 disrupted angiogenesis in the Calu6 model, we examinedthe vasculature in representative tumor sections from mice in the threetreatment groups. By normalizing staining of CD31, an endothelial cellmarker, to the staining of DNA, using DAPI, we found that anti-NRR1significantly increased CD31 staining relative to the other groups (FIG.14D). This increase was statistically significant (p<0.01), and wasconsistently observed over multiple images (FIG. 14E). In contrast andas expected, anti-VEGF caused a reduction in CD31 staining (FIGS. 14Dand 14E). The results from selective blocking of Notch1 thus mirrorthose reported for selective blocking of Dll4, which similarly leads toan increase in tumor CD31 staining as well as the generation of poorlyfunctioning tumor vasculature. Taken together, these results suggestthat the anti-tumor effects exerted by anti-NRR1 in the Calu6 and HM7models primarily reflect a disruption in tumor angiogenesis.

8. Antibody Inhibition of Both Notch1 and Notch2 has Marked Effects onIntestinal Cell Fate

Gamma-secretase inhibitors (GSIs), which are pan-Notch inhibitors thatinhibit multiple Notch receptors, cause weight loss and intestinalgoblet cell metaplasia, reflecting the role that Notch plays indetermining cell fate by maintaining proliferation of intestinal cryptprogenitor cells and prohibiting differentiation to a secretory cellfate. (See van Es et al., Nature 435:959-963 (2005).) Moreover, geneticstudies using conditional Notch knockout mice suggest that disruption ofboth Notch1 and Notch2 is required to cause intestinal goblet cellmetaplasia. (See Riccio et al., EMBO Rep. 9:377-383 (2008).) We usedanti-NRR1 and anti-NRR2 to determine the effects of selective inhibitionof Notch1 or Notch2, or both, on weight loss and intestinal celldifferentiation.

Mice treated with anti-NRR1 displayed a mild decrease in overall bodyweight during the course of antibody dosing. In a first experimentdirectly comparing anti-NRR1 and anti-NRR2, we found that anti-NRR1caused a 5% decrease in total body weight, in contrast to the weightgains observed in the anti-NRR2 as well as the anti-gD and anti-LTβR-Fccontrol groups (FIG. 15A). In a second experiment, selective inhibitionof Notch1 or Notch2 using anti-NRR1 or anti-NRR2, respectively, resultedin little or no effect on weight. The results of that experiment aredepicted in FIG. 24A, in which mice were dosed with 5 mg/kg anti-NRR1(squares), anti-NRR2 (triangles), or a control antibody (“α-gD”,diamonds) on the days indicated by arrows. However, mice treated withboth anti-NRR1 and anti-NRR2 (“X”s in FIG. 24A) lost nearly 20% of theirstarting body weight by as early as day 7.

We investigated whether the subtle weight loss resulting from anti-NRR1treatment alone, or the more dramatic weight loss resulting fromtreatment with both anti-NRR1 and anti-NRR2, reflected changes inintestinal cell fate determination. After dosing mice with anti-NRR1 oranti-gD or DBZ as controls, sections of the large (FIG. 19) and smallintestines were analyzed at day 2 (FIG. 20) and day 7 (FIG. 15B) using avariety of histochemical stains. DBZ had the same effects as previouslydescribed for GSIs (FIG. 15B, DBZ column): inhibition of Hes1 expressioncorrelated with a decrease in progenitor cell proliferation (as shown byKi-67 staining) and a dramatic expansion of the goblet cell population(as shown by Alcian Blue staining of mucin). Lysozyme staining alsosuggested that DBZ increased the numbers and/or activity of Panethcells, a second secretory cell population (FIG. 15B), a suggestion thatwas further supported using Azure A-Eosin B staining for Paneth cellgranules (data not shown). Anti-NRR1 at 10 mg/kg induced intestinalchanges indistinguishable from those induced by DBZ (FIG. 15A, compareDBZ 30 μmol/kg to α-NRR1 10 mg/kg). These intestinal changes depended onthe concentration of anti-NRR1, such that significant cell fate changeswere observed using 10 mg/kg and 2.0 mg/kg but few, if any, notablechanges were observed using 0.4 mg/kg. The large intestine respondedsimilarly to DBZ and anti-NRR1 treatment: both treatments significantlyreduced Notch signaling (FIG. 19, Hes1 staining), blocked progenitorcell proliferation (FIG. 19, Ki-67) and increased goblet cell numbers(FIG. 19, Alcian Blue and H&E). The intestinal cell fate changes wereonly weakly detectable at an early time point, day 2 of this study (FIG.20; note the slight but detectable changes, caused by both DBZ andanti-NRR1, observed in Ki-67 and Alcian Blue staining).

To determine whether inhibition of Notch2 signaling might similarlyaffect intestinal cell fate determination, we directly compared theeffects of anti-NRR1 and anti-NRR2 on the large (data not shown) andsmall intestines (FIG. 15C, showing Alcian Blue and Ki-67 staining, andFIG. 24B, showing Alcian Blue staining). Whereas anti-NRR1 caused gobletcell metaplasia that coincided with a decrease in Ki-67 expression inthe crypt progenitors, anti-NRR2 did not cause any detectable effects(FIGS. 15C and 24B, compare anti-NRR2 to anti-gD control). Becauseanti-NRR1 and anti-NRR2 function as potent and specific inhibitors ofNotch1 and Notch2 signaling, respectively, both in vivo (FIG. 11) and invitro (FIGS. 9, 10 and 21), these results strongly suggest thatselective inhibition of Notch1, but not Notch2, is sufficient to alterintestinal cell fate determination.

The effect of inhibiting both Notch1 and Notch2 on intestinal cell fatedetermination was also investigated. FIG. 22 shows synergistic effectsof anti-NRR1 and anti-NRR2 on altered intestinal cell differentiation.Female Balb/c mice were dosed with 5 mg/kg anti-NRR1, 5 mg/kg anti-NRR2or 5 mg/kg anti-NRR1 plus 5 mg/kg anti-NRR2 on days 0, 4, 7 and 11. Theintestines were harvested and stained with H&E to reveal the cryptmorphology. For mice treated with anti-NRR1 or anti-NRR2 alone, theintestines were harvested on day 12; for mice treated with bothantibodies together, the intestines were harvested on day 6 because themice in this group were rapidly losing weight. The analysis of theintestines indicated that the antibody combination resulted in a moresevere phenotype than that observed following treatment with anti-NRR1alone, suggesting that Notch1 and Notch2 function together and partiallyredundantly in the mammalian intestine. Further experimentationconfirmed this observation. As shown in FIG. 24B, intestines from micetreated with anti-NRR1 and anti-NRR2 showed severe goblet cellmetaplasia. Although treatment with anti-NRR1 alone was sufficient toinduce some goblet cell metaplasia, the effect of anti-NRR1 alone wasmild relative to the effect of both anti-NRR1 and anti-NRR2 incombination.

Taken together, the results indicate that Notch1 and Notch2 functionredundantly in intestinal cell differentiation, although potentinhibition of Notch1 but not Notch2 is sufficient to reveal a partialphenotype. We hypothesize that our ability to detect this partialphenotype, not previously reported in genetic studies, reflects thatanti-NRR1 provides a more uniform and potent inhibition of Notch1signaling throughout cells of the crypt than can be achieved followingconditional gene knockout, which may be incomplete. Importantly, bysignificantly reducing or avoiding the goblet cell metaplasia that is ahallmark of general Notch inhibition, the Notch1- and Notch2-specificantibody inhibitors reported herein represent a clear breakthrough overexisting pan-Notch inhibitors such as GSIs.

9. Rescue of Anti-NRR1 Intestinal Phenotype

FIG. 23 shows that dexamethasone at least partially rescues theintestinal phenotype caused by anti-NRR1. Antibodies were administeredintraperitoneally (IP) into female NCR.nude mice (three mice per group)as follows:

-   -   Group A) Vehicle combination, MCT, once daily, anti-gD control        antibody at 4 mg/kg, every fourth day (not shown in figure);    -   Group B) Dexamethasone, 90 mg/kg, daily (not shown in figure);    -   Group C) Anti-NRR1, 4 mg/kg, every fourth day;    -   Group D) Dexamethasone, 90 mg/kg, daily, and Anti-NRR1, 4 mg/kg,        every fourth day.

The vehicle was 0.5% (w/v) hydroxypropylmethylcellulose (Methocel E4M)and 0.1% (w/v) Tween 80 in water (MCT), previously shown not to causeadverse clinical effects.

Intestines were harvested on day 9 and stained with anti-Ki67(proliferation marker) and alcian blue (for mucin). This experimentrevealed that mice treated with the dexamethasone plus anti-NRR1combination displayed a milder intestinal phenotype relative to micetreated with anti-NRR1 alone, thus suggesting that dexamethasoneprotects the intestine from anti-NRR1-induced changes indifferentiation.

10. Anti-NRR2 Suppresses Growth of Melanoma Cell Lines

The effect of anti-NRR1 and anti-NRR2 on the growth of melanoma celllines was investigated. Human melanoma cell lines SK23 and LOX-IMVI wereplated in medium containing 2% FBS at low density (4000 cells/well forSK23, 2500 cells/well for LOX-IMVI) in 96 well plates that were coatedwithout (−jag-1) or with Jagged-1 ligand (+jag-1) (R&D Systems,Minneapolis, Minn.). Anti-NRR1 (20 mg/ml), anti-NRR2 (20 mg/ml), orgamma-secretase inhibitor (GSI) DAPT (5 μM) was added to the culturesand re-added every other day thereafter. An equal volume of DMSO servedas the vehicle control, whereas an equal concentration of anti-gD HuB6phage antibody served as the isotype control antibody. Cell Titer Gloassays (Promega, Madison, Wis.) were performed six days after plating.The results are shown in FIGS. 25A and 25B (anti-NRR1 and anti-NRR2 arereferred to as “anti-N1” and “anti-N2”). In FIGS. 25A and 25B, cellviability (y-axis) is expressed relative to the values from the DMSOvehicle, −jag-1 control wells. Both melanoma cell lines showed adecrease in viability in response to GSI treatment (especially in thepresence of Jag1 ligand) and anti-NRR2 treatment, but not anti-NRR1treatment.

11. Anti-NRR2 Suppresses Growth of Diffuse Large B-Cell Lymphoma InVitro

Diffuse large B-cell lymphoma (DLBCL) is the most common type ofnon-Hodgkin's lymphoma. A recent study of cancer cells from patientswith DLBCL indicated that approximately 8% of patients carry a PESTdomain mutation in Notch2, a mutation predicted to prolong Notch2signaling following ligand activation. (See Lee et al., Cancer Sci.100:920-926, 2009.) However, how DLBCL cells would respond to Notch2inhibition was not known. Therefore, we investigated the effect ofanti-NRR2 on five DLBCL cell lines. The DLBCL lines listed on the rightof FIG. 26 were grown in replicates for three days in wells of a384-well plate. The cell cultures included the indicated concentrationsof anti-NRR2 (referred to as “anti-Notch2” in FIGS. 26 and 27), whichcorresponded to three-fold serial dilutions starting at 10 μg/ml. Growthwas assessed using Cell Titer Glo assays (Promega), plotted as thepercentage of cell viability relative to treatment with an isotypecontrol antibody. The data points in FIG. 26 represent average valuesobtained from individual wells for each cell line at the indicatedantibody concentrations. As shown in FIG. 26, growth of one of the celllines, “DB,” was strongly inhibited by treatment with anti-NRR2. FIG. 27shows growth of the DB cell line over time. The DB line was grown inwells of a 12-well plate, and the cultures were treated with DMSO, thegamma-secretase inhibitor DAPT, anti-gD isotype control antibody oranti-NRR2 at the indicated concentrations. Growth was assessed bycounting viable cells (Vi-CELL, Beckman Coulter, Fullerton, Calif.) atdays 2, 3 and 5 after inoculating and treating the cultures. Valuesrepresent the mean of measurements from three independent cultures,plus/minus the standard deviation. FIG. 27 shows that anti-NRR2suppresses the growth of the DB DLBCL cell line in vitro.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literatures cited herein are expressly incorporated in theirentirety by reference.

1. A monoclonal antibody that binds to Notch2 NRR and inhibits Notch2activity, wherein the antibody binds to mouse Notch2 NRR and humanNotch2 NRR.
 2. The antibody of claim 1, wherein the antibody does notsignificantly bind to a Notch family member other than Notch2. 3.(canceled)
 4. (canceled)
 5. The antibody of claim 1, wherein theantibody comprises: (a) an HVR-H1 comprising an amino acid sequence thatconforms to the consensus sequence of SEQ ID NO:3; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:4; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:5; (d) an HVR-L1comprising an amino acid sequence that conforms to the consensussequence of SEQ ID NO:10; (e) an HVR-L2 comprising an amino acidsequence that conforms to the consensus sequence of SEQ ID NO:14; and(f) an HVR-L3 comprising an amino acid sequence that conforms to theconsensus sequence of SEQ ID NO:19.
 6. The antibody of claim 5, whereinthe antibody comprises an HVR-H1 comprising an amino acid sequenceselected from SEQ ID NOs:1-2; an HVR-L1 comprising an amino acidsequence selected from SEQ ID NOs:6-9; an HVR-L2 comprising an aminoacid sequence selected from SEQ ID NOs:11-13; and an HVR-L3 comprisingan amino acid sequence selected from SEQ ID NOs:15-18.
 7. The antibodyof claim 6, wherein the HVR-H1 comprises the amino acid sequence of SEQID NO:1, the HVR-L1 comprises the amino acid sequence of SEQ ID NO:6,the HVR-L2 comprises the amino acid sequence of SEQ ID NO:11, and theHVR-L3 comprises the amino acid sequence of SEQ ID NO:15.
 8. Theantibody of claim 6, wherein the HVR-H1 comprises the amino acidsequence of SEQ ID NO:2, the HVR-L1 comprises the amino acid sequence ofSEQ ID NO:7, the HVR-L2 comprises the amino acid sequence of SEQ IDNO:11, and the HVR-L3 comprises the amino acid sequence of SEQ ID NO:16.9. The antibody of claim 6, wherein the HVR-H1 comprises the amino acidsequence of SEQ ID NO:2, the HVR-L1 comprises the amino acid sequence ofSEQ ID NO:8, the HVR-L2 comprises the amino acid sequence of SEQ IDNO:12, and the HVR-L3 comprises the amino acid sequence of SEQ ID NO:17.10. The antibody of claim 6, wherein the HVR-H1 comprises the amino acidsequence of SEQ ID NO:2, the HVR-L1 comprises the amino acid sequence ofSEQ ID NO:9, the HVR-L2 comprises the amino acid sequence of SEQ IDNO:13, and the HVR-L3 comprises the amino acid sequence of SEQ ID NO:18.11. The antibody of claim 5, further comprising at least one frameworkselected from a human VH Acceptor 2 framework and a human VL kappasubgroup I consensus framework.
 12. The antibody of claim 1, wherein theantibody comprises a heavy chain variable domain having at least 90%sequence identity to an amino acid sequence selected from SEQ IDNO:20-21 and a light chain variable domain having at least 90% sequenceidentity to an amino acid sequence selected from SEQ ID NO:22-25. 13.The antibody of claim 12, wherein the antibody comprises a heavy chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:20 and a light chain variable domain having atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:22.14. The antibody of claim 13, wherein the heavy chain variable domaincomprises the amino acid sequence of SEQ ID NO:20, and the light chainvariable domain comprises the amino acid sequence of SEQ ID NO:22. 15.The antibody of claim 12, wherein the antibody comprises a heavy chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:21 and a light chain variable domain having atleast 90% sequence identity to an amino acid sequence selected from SEQID NOs:23-25.
 16. The antibody of claim 15, wherein the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO:21, andthe light chain variable domain comprises an amino acid sequenceselected from SEQ ID NOs:23-25.
 17. (canceled)
 18. (canceled)
 19. Theantibody of claim 1, wherein the antibody is an antibody fragmentselected from a Fab, Fab′-SH, Fv, scFv, or (Fab′)2 fragment.
 20. Theantibody of claim 1, wherein the antibody is human, humanized, orchimeric.
 21. A method of inhibiting Notch2 activity, the methodcomprising exposing a cell that expresses Notch2 to the antibody ofclaim
 1. 22. A method of treating a disorder associated with increasedexpression or activity of Notch2, the method comprising administering toa subject in need thereof an effective amount of the antibody ofclaim
 1. 23. A method of treating a B-cell malignancy, the methodcomprising administering to a subject in need thereof an effectiveamount of the antibody of claim
 1. 24. A method of treating melanoma,the method comprising administering to a subject in need thereof aneffective amount of the antibody of claim 1.